Variant maltohexaose-forming alpha-amylase variants

ABSTRACT

Disclosed are compositions and methods relating to variant maltohexaose-forming alpha-amylases. The variant alpha-amylases are useful, for example, for starch liquefaction and saccharification, for cleaning starchy stains in laundry, dishwashing, and other applications, for textile processing (e.g., desizing), in animal feed for improving digestibility, and for baking and brewing.

PRIORITY

The present application is a continuation of U.S. patent applicationSer. No. 14/354,425, filed Apr. 25, 2014, which is a U.S. National PhaseApplication of International Application No. PCT/US2012/062209, filedOct. 26, 2012, which claims priority to U.S. Provisional ApplicationSer. No. 61/552,910, filed on Oct. 28, 2011, and U.S. ProvisionalApplication Ser. No. 61/668,359, filed on Jul. 5, 2012, which are herebyincorporated by reference in their entirety.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 C.F.R. §1.52(e), is incorporated herein by reference. The sequence listing textfile submitted via EFS contains the file “NB40279USCNT-SEQ-LIST.txt”created on May 18, 2016, which is 20,480 bytes in size.

FIELD OF THE INVENTION

Disclosed are compositions and methods relating to variantmaltohexaose-forming α-amylases. The variant α-amylases are useful, forexample, for starch liquefaction and saccharification, cleaning starchystains, textile desizing, baking, and brewing.

BACKGROUND

Starch consists of a mixture of amylose (15-30% w/w) and amylopectin(70-85% w/w). Amylose consists of linear chains of α-1,4-linked glucoseunits having a molecular weight (MW) from about 60,000 to about 800,000.Amylopectin is a branched polymer containing α-1,6 branch points every24-30 glucose units; its MW may be as high as 100 million.

Sugars from starch, in the form of concentrated dextrose syrups, arecurrently produced by an enzyme catalyzed process involving: (1)liquefaction (or viscosity reduction) of solid starch with an α-amylaseinto dextrins having an average degree of polymerization of about 7-10,and (2) saccharification of the resulting liquefied starch (i.e. starchhydrolysate) with amyloglucosidase (also called glucoamylase or GA). Theresulting syrup has a high glucose content. Much of the glucose syrupthat is commercially produced is subsequently enzymatically isomerizedto a dextrose/fructose mixture known as isosyrup. The resulting syrupalso may be fermented with microorganisms, such as yeast, to producecommercial products including ethanol, citric acid, lactic acid,succinic acid, itaconic acid, monosodium glutamate, gluconates, lysine,other organic acids, other amino acids, and other biochemicals, forexample. Fermentation and saccharification can be conductedsimultaneously (i.e., an SSF process) to achieve greater economy andefficiency.

α-amylases hydrolyze starch, glycogen, and related polysaccharides bycleaving internal α-1,4-glucosidic bonds at random. α-amylases,particularly from Bacilli, have been used for a variety of differentpurposes, including starch liquefaction and saccharification, textiledesizing, starch modification in the paper and pulp industry, brewing,baking, production of syrups for the food industry, production offeedstocks for fermentation processes, and in animal feed to increasedigestability. These enzymes can also be used to remove starchy soilsand stains during dishwashing and laundry washing.

SUMMARY

The present compositions and methods relate to variantmaltohexaose-forming amylase polypeptides, and methods of use, thereof.Aspects and embodiments of the present compositions and methods aresummarized in the following separately-numbered paragraphs:

1. In one aspect, a variant α-amylase polypeptide derived from aparental α-amylase polypeptide is provided, comprising at least onecombinable mutation at a productive amino acid position; wherein: (i)the combinable mutation is the substitution of an amino acid residuepresent in the parental α-amylase with a different amino acid residue,which improves at least one desirable property of the variant α-amylasecompared to the parental α-amylase, while not significantly decreasingeither expression, activity, or stability of the variant α-amylase,compared to the parental α-amylase, (ii) the productive position is anamino acid position that can be substituted with a plurality ofdifferent amino acid residues, each of which substitutions result in avariant α-amylase that meets the requirements of (i), and (iii) thecombinable mutation corresponds to a mutation listed in Lists A, B, C,or D, or in Table C or D, which use SEQ ID NO: 3 for numbering.

2. In some embodiments of the variant amylase of paragraph 1, each ofthe at least one combinable mutations produces a variant amylase whereinthe minimum performance indices (PI) relative to the parental amylasefor (i) protein expression, (ii) activity, and (iii) detergent stabilityor thermostability are greater than or equal to 0.9, and the PI for anyone of (i), (ii), or (iii) that is greater than or equal to 1.0.

3. In some embodiments of the variant amylase of paragraph 1, each ofthe at least one combinable mutations produces a variant amylase whereinthe minimum performance indices (PI) relative to the parental amylasefor (i) protein expression, (ii) activity, and (iii) detergent stabilityor thermostability are greater than or equal to 0.8, and the PI for anyone of (i), (ii), or (iii) that is greater than or equal to 1.2.

4. In some embodiments of the variant amylase of paragraph 1, each ofthe at least one combinable mutations produces a variant amylase whereinthe minimum performance indices (PI) relative to the parental amylasefor (i) protein expression, (ii) activity, and (iii) detergent stabilityor thermostability are greater than or equal to 0.5, and the PI for anyone of (i), (ii), or (iii) that is greater than or equal to 1.5.

5. In some embodiments of the variant amylase of any of the precedingparagraphs, each of the at least one combinable mutations have asuitability score of +++, ++++, or +++++, referring to Table B.

6. In some embodiments of the variant amylase of any of the precedingparagraphs, each of the at least one combinable mutation have asuitability score of ++++, or +++++, referring to Table B.

7. In some embodiments of the variant amylase of any of the precedingparagraphs, each of the at least one combinable mutation has asuitability score of +++++, referring to Table B.

8. In some embodiments of the variant amylase of any of the precedingparagraphs, each of the at least one combinable mutation has aproductivity score of 1 or 2.

9. In some embodiments, the variant amylase of any of the precedingparagraphs includes a plurality of combinable mutations.

10. In some embodiments, the variant amylase of any of the precedingparagraphs further comprises a deletion corresponding to a residueselected from the group consisting of Arg-181, Gly-182, His-183, andGly-184, using SEQ ID NO: 3 for numbering.

11. In some embodiments, the variant amylase of any of the precedingparagraphs further comprises deletions corresponding to residues Arg-181and Gly-182, using SEQ ID NO: 3 for numbering.

12. In some embodiments of the variant amylase of any of the precedingparagraphs, the parental α-amylase or the variant α-amylase has at least60% amino acid sequence identity to the amino acid sequence of SEQ IDNO: 3 or SEQ ID NO: 4, or wherein the parental α-amylase or the variantα-amylase is encoded by a nucleic acid that hybridizes under stringentconditions to the nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 5.

13. In some embodiments of the variant amylase of any of the precedingparagraphs, the parental α-amylase or the variant α-amylase has at least70% amino acid sequence identity to the amino acid sequence of SEQ IDNO: 3 or SEQ ID NO: 4, or wherein the parental α-amylase or the variantα-amylase is encoded by a nucleic acid that hybridizes under stringentconditions to the nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 5.

14. In some embodiments of the variant amylase of any of the precedingparagraphs, the parental α-amylase or the variant α-amylase has at least80% amino acid sequence identity to the amino acid sequence of SEQ IDNO: 3 or SEQ ID NO: 4, or wherein the parental α-amylase or the variantα-amylase is encoded by a nucleic acid that hybridizes under stringentconditions to the nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 5.

15. In some embodiments of the variant amylase of any of the precedingparagraphs, the parental α-amylase or the variant α-amylase has at least90% amino acid sequence identity to the amino acid sequence of SEQ IDNO: 3 or SEQ ID NO: 4, or wherein the parental α-amylase or the variantα-amylase is encoded by a nucleic acid that hybridizes under stringentconditions to the nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 5.

16. In another aspect, a composition comprising the variant amylase ofany of paragraphs 1-15 is provided.

17. In some embodiments of the composition of paragraph 16, thecomposition is effective for removing starchy stains from laundry,dishes, or textiles.

18. In some embodiments, the composition of paragraphs 16 or 17 furthercomprises a surfactant.

19. In some embodiments of the composition of paragraphs 16-18, thecomposition is a detergent composition.

20. In some embodiments of the composition of paragraphs 16-19, thecomposition is a laundry detergent or a laundry detergent additive.

21. In some embodiments of the composition of paragraphs 16-20, thecomposition is a manual or automatic dishwashing detergent.

22. In some embodiments, the composition of paragraphs 16-21 furthercomprises one or more additional enzymes selected from the groupconsiting of protease, hemicellulase, cellulase, peroxidase, lipolyticenzyme, metallolipolytic enzyme, xylanase, lipase, phospholipase,esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase,keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase,pullulanase, tannase, pentosanase, malanase, β-glucanase, arabinosidase,hyaluronidase, chondroitinase, laccase, and an amylase other than theamylase of any one of paragraphs 1-15.

23. In some embodiments, the composition of paragraph 16 is forliquifying starch.

24. In some embodiments, the composition of paragraph 16 is forsaccharifying a composition comprising starch, for SSF postliquefaction, or for direct SSF without prior liquefaction.

25. In some embodiments, the composition of paragraph 16 is forproducing a fermented beverage.

26. In some embodiments, the composition of paragraph 16 is forproducing a baked food product.

27. In some embodiments, the composition of paragraph 16 is for textiledesizing.

28. In another aspect, a method for removing a starchy stain or soilfrom a surface is provided, comprising: contacting the surface in thepresence of a aqueous composition comprising an effective amount of thevariant amylase of any of the paragraphs 1-15 and, allowing thepolypeptide to hydrolyze starch components present in the starchy stainto produce smaller starch-derived molecules that dissolve in the aqueouscomposition, and rinsing the surface, thereby removing the starchy stainfrom the surface.

29. In some embodiments of the method of paragraph 28, the aqueouscomposition further comprises a surfactant.

30. In some embodiments of the method of paragraphs 28-29, the surfaceis a textile surface.

31. In some embodiments of the method of paragraphs 28-29, the surfaceis on dishes.

32. In some embodiments of the method of paragraphs 28-29, the surfaceis a soiled hard surface.

33. In some embodiments of the method of paragraphs 28-32, thecomposition further comprises at least one additional enzymes selectedfrom the group consiting of protease, hemicellulase, cellulase,peroxidase, lipolytic enzyme, metallolipolytic enzyme, xylanase, lipase,phospholipase, esterase, perhydrolase, cutinase, pectinase, pectatelyase, mannanase, keratinase, reductase, oxidase, phenoloxidase,lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase,β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, andan amylase other than the amylase of any one of paragraphs 1-15.

34. In another aspect, a method of saccharifying a compositioncomprising starch to produce a composition comprising glucose isprovided, wherein the method comprises: (i) contacting the solutioncomprising starch with effective amount of the variant amylase of any ofthe paragraphs 1-15; and (ii) saccharifying the solution comprisingstarch to produce the composition comprising glucose; wherein thevariant amylase catalyzes the saccharification of the starch solution toglucose.

35. In some embodiments of the method of paragraph 34, the compositioncomprising starch comprises liquefied starch, gelatinized starch, orgranular starch.

36. In some embodiments of the method of paragraphs 34 or 35,saccharification is conducted at a temperature range of about 30° C. toabout 75° C.

37. In some embodiments of the method of paragraph 36, the temperaturerange is 47° C.-74° C.

38. In some embodiments of the method of any of paragraphs 34-37,saccharification is conducted over a pH range of pH 2.0-7.5.

39. In some embodiments of the method of paragraph 38, the pH range ispH 3.5-5.5.

40. In some embodiments of the method of paragraph 39, the pH range ispH 3.5-4.5.

41. In some embodiments, the method of any of paragraphs 34-40, furthercomprises fermenting the glucose composition to produce an end offermentation (EOF) product.

42. In some embodiments of the method of paragraph 41, the fermentationis a simultaneous saccharification and fermentation (SSF) reaction.

43. In some embodiments of the method of paragraphs 41 or 42, thefermentation is conducted for 48-70 hours at pH 2-8 and in a temperaturerange of 25° C.-70° C.

44. In some embodiments of the method of any of paragraphs 41-43, theEOF product comprises ethanol.

45. In some embodiments of the method of any of paragraphs 41-44, theEOF product comprises 8-18% (v/v) ethanol.

46. In some embodiments of the method of paragraph s41-45, the methodfurther comprises contacting a mash and/or a wort with an amylase.

47. In some embodiments of the method of paragraph 46, the methodfurther comprises: (a) preparing a mash; (b) filtering the mash toobtain a wort; and (c) fermenting the wort to obtain a fermentedbeverage, wherein the variant amylase of any one of paragraphs 1-16 and74-80 is added to: (i) the mash of step (a) and/or (ii) the wort of step(b) and/or (iii) the wort of step (c).

48. In some embodiments of the method of any of paragraphs 41-47, theEOF product comprises a metabolite.

49. In some embodiments of the method of paragraph 48, the metabolite iscitric acid, lactic acid, succinic acid, monosodium glutamate, gluconicacid, sodium gluconate, calcium gluconate, potassium gluconate, gluconodelta-lactone, sodium erythorbate, omega 3 fatty acid, butanol, an aminoacid, lysine, itaconic acid, 1,3-propanediol, or isoprene.

50. In some embodiments, the method of any of paragraphs 34-49 furthercomprises adding glucoamylase, hexokinase, xylanase, glucose isomerase,xylose isomerase, phosphatase, phytase, pullulanase, β-amylase,α-amylase that is not the variant α-amylase, protease, cellulase,hemicellulase, lipase, cutinase, isoamylase, redox enzyme, esterase,transferase, pectinase, alpha-glucosidase, beta-glucosidase, or acombination thereof, to the starch solution.

51. In some embodiments of the method of paragraph 50, the glucoamylaseis added to 0.1-2 glucoamylase units (GAU)/g ds.

52. In some embodiments of the method of any of paragraphs 34-51, theamylase is expressed and secreted by a host cell.

53. In some embodiments of the method of paragraph 52, the compositioncomprising starch is contacted with the host cell.

54. In some embodiments of the method of paragraphs 52 or 53, the hostcell further expresses and secretes a glucoamylase or other enzyme.

55. In some embodiments of the method of any of paragraphs 52-54, thehost cell is capable of fermenting the composition.

56. In another aspect, a composition comprising glucose produced by themethod of any one of paragraphs 34-55 is provided.

57. In another aspect, a liquefied starch produced by the method of anyone of paragraphs 34-55 is provided.

58. In another aspect, a fermented beverage produced by the method ofany one of paragraphs 34-55 is provided.

59. In another aspect, the use of an amylase of any of paragraphs 1-15in the production of a composition comprising glucose is provided.

60. In another aspect, the use of an amylase of any of paragraphs 1-15in the production of a liquefied starch is provided.

61. In another aspect, the use of an amylase of any of paragraphs 1-15in the production of a fermented beverage is provided.

62. In another aspect, the use of an amylase of any of paragraphs 1-15in cleaning starchy stains is provided.

63. In another aspect, the use of an amylase of any of paragraphs 1-15in textile desizing is provided.

64. In some embodiments of the method according to any one of paragraphs34-55, the fermented beverage of paragraph 58, or the use of paragraph61, the fermented beverage or end of fermentation product is selectedfrom the group consisting of (i) a beer selected from the groupconsisting of full malted beer, beer brewed under the “Reinheitsgebot”,ale, IPA, lager, bitter, Happoshu (second beer), third beer, dry beer,near beer, light beer, low alcohol beer, low calorie beer, porter, bockbeer, stout, malt liquor, non-alcoholic beer, and non-alcoholic maltliquor; and (ii) cereal or malt beverages selected from the groupconsisting of fruit flavoured malt beverages, liquor flavoured maltbeverages, and coffee flavoured malt beverages.

65. In another aspect, a method of producing a food composition isprovided, comprising combining: (i) one or more food ingredients, and(ii) a variant α-amylase of any of paragraphs 1-15, wherein the variantα-amylase thereof catalyzes the hydrolysis of starch components presentin the food ingredients to produce glucose.

66. In some embodiments of the method of paragraph 65, the foodcomposition is selected from the group consisting of a food product, abaking composition, a food additive, an animal food product, a feedproduct, a feed additive, an oil, a meat, and a lard.

67. In some embodiments of the method of any one of paragraphs 65-66,the one or more food ingredients comprise a baking ingredient or anadditive.

68. In some embodiments of the method of any one of paragraphs 65-67,the one or more food ingredients is/are selected from the groupconsisting of flour; an anti-staling amylase; a phospholipase; aphospholipid; a maltogenic alpha-amylase or a variant, homologue, ormutants thereof which has maltogenic alpha-amylase activity; a bakeryxylanase; and a lipase.

69. In some embodiments of the method of paragraph 65, the one or morefood ingredients is/are selected from the group consisting of: (i) amaltogenic alpha-amylase from Bacillus stearothermophilus, (ii) a bakeryxylanase is from Bacillus, Aspergillus, Thermomyces or Trichoderma,(iii) a glycolipase from Fusarium heterosporum.

70. In some embodiments of the method of any one of paragraphs 65-69,the food composition comprises a dough or a dough product, preferably aprocessed dough product.

71. In some embodiments, the method of any one of paragraphs 65-70further comprises baking the food composition to produce a baked good.

72. In some embodiments, the method of any one of paragraphs 65-70,further comprises: (i) providing a starch medium; (ii) adding to thestarch medium an amylase; and (iii) applying heat to the starch mediumduring or after step (b) to produce a bakery product.

73. In another aspect, a method of desizing a textile is provided,comprising contacting a desizing composition with a sized textile for atime sufficient to desize the textile, wherein the desizing compositioncomprises a variant α-amylase of any one of paragraphs 1-15.

74. In another aspect, an isolated polynucleotide encoding a polypeptideof any of paragraphs 1-15 is provided.

75. In another aspect, an expression vector comprising thepolynucleotide of paragraph 74 is provided.

76. In another aspect, a host cell comprising the expression vector ofparagraph 75 is provided.

These and other aspects and embodiments of the compositions and methodswill be apparent from the present description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Clustal alignment, using default parameters, of Amy707amylase and AA560 amylase.

FIG. 2 is a map of the pHPLT vector comprising the Amy707 gene(pHPLT-Amy707).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 sets forth a codon-modified nucleotide sequence in theplasmid pHPLT-Amy707 that encodes the mature form of Bacillus sp. 707α-amylase. The sequence encoding the LAT signal peptide is underlined.

SEQ ID NO: 2 sets forth the amino acid sequence of the precursor form ofBacillus sp. 707 α-amylase produced from the plasmid pHPLT-Amy707. TheLAT signal peptide is underlined.

SEQ ID NO: 3 sets forth the amino acid sequence of the mature form ofBacillus sp. 707 α-amylase produced from the plasmid pHPLT-Amy707.

SEQ ID NO: 4 sets forth the amino acid sequence of the mature form ofAA560 α-amylase derived from Bacillus sp. DSM 12649 (i.e., the parent ofSTAINZYME™).

SEQ ID NO: 5 sets forth Genebank Accession No. M18862, which encodesBacillus sp. 707 α-amylase.

DETAILED DESCRIPTION

Described are compositions and methods relating to variantmaltohexaose-forming amylase enzymes. The variants were discovered by acombination of experimental approaches, as detailed in the appendedExamples. The approaches include the use of site evaluation libraries(SELs) and structure-based analysis. Exemplary applications for thevariant amylase enzymes are for starch liquefaction andsaccharification, for cleaning starchy stains in laundry, dishwashing,and other applications, for textile processing (e.g., desizing), inanimal feed for improving digestibility, and for baking and brewing.These and other aspects of the compositions and methods are described indetail, below.

Prior to describing the various aspects and embodiments of the presentcompositions and methods, the following definitions and abbreviationsare described.

1. Definitions and Abbreviations

In accordance with this detailed description, the followingabbreviations and definitions apply. Note that the singular forms “a,”“an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an enzyme” includesa plurality of such enzymes, and reference to “the dosage” includesreference to one or more dosages and equivalents thereof known to thoseskilled in the art, and so forth.

The present document is organized into a number of sections for ease ofreading; however, the reader will appreciate that statements made in onesection may apply to other sections. In this manner, the headings usedfor different sections of the disclosure should not be construed aslimiting.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. The following terms are provided below.

1.1. Abbreviations and Acronyms

The following abbreviations/acronyms have the following meanings unlessotherwise specified:

-   -   ABTS 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid    -   AE or AEO alcohol ethoxylate    -   AES or AEOS alcohol ethoxysulfate    -   AkAA Aspergillus kawachii α-amylase    -   AnGA Aspergillus niger glucoamylase    -   AOS α-olefinsulfonate    -   AS alkyl sulfate    -   cDNA complementary DNA    -   CMC carboxymethylcellulose    -   DE dextrose equivalent    -   DNA deoxyribonucleic acid    -   DPn degree of saccharide polymerization having n subunits    -   ds or DS dry solids    -   DTMPA diethylenetriaminepentaacetic acid    -   EC Enzyme Commission    -   EDTA ethylenediaminetetraacetic acid    -   EO ethylene oxide (polymer fragment)    -   EOF End of Fermentation    -   GA glucoamylase    -   GAU/g ds glucoamylase activity unit/gram dry solids    -   HFCS high fructose corn syrup    -   HgGA Humicola grisea glucoamylase    -   IPTG isopropyl β-D-thiogalactoside    -   IRS insoluble residual starch    -   kDa kiloDalton    -   LAS linear alkylbenzenesulfonate    -   LAT, BLA B. lichenifonnis amylase    -   MW molecular weight    -   MWU modified Wohlgemuth unit; 1.6×10⁻⁵ mg/MWU=unit of activity    -   NCBI National Center for Biotechnology Information    -   NOBS nonanoyloxybenzenesulfonate    -   NTA nitriloacetic acid    -   OxAm Purastar HPAM 5000L (Danisco US Inc.)    -   PAHBAH p-hydroxybenzoic acid hydrazide    -   PEG polyethyleneglycol    -   pI isoelectric point    -   PI performance index    -   ppm parts per million, e.g., μg protein per gram dry solid    -   PVA poly(vinyl alcohol)    -   PVP poly(vinylpyrrolidone)    -   RCF relative centrifugal/centripetal force (i.e., x gravity)    -   RNA ribonucleic acid    -   SAS alkanesulfonate    -   SDS-PAGE sodium dodecyl sulfate polyacrylamide gel        electrophoresis    -   SSF simultaneous saccharification and fermentation    -   SSU/g solid soluble starch unit/gram dry solids    -   sp. species    -   TAED tetraacetylethylenediamine    -   Tm melting temperature    -   TrGA Trichoderma reesei glucoamylase    -   w/v weight/volume    -   w/w weight/weight    -   v/v volume/volume    -   wt % weight percent    -   ° C. degrees Centigrade    -   H₂O water    -   dH₂O or DI deionized water    -   dIH₂O deionized water, Milli-Q filtration    -   g or gm grams    -   micrograms    -   mg milligrams    -   kg kilograms    -   μL and μl microliters    -   mL and ml milliliters    -   mm millimeters    -   μm micrometer    -   M molar    -   mM millimolar    -   micromolar    -   U units    -   sec seconds    -   min(s) minute/minutes    -   hr(s) hour/hours    -   DO dissolved oxygen    -   Ncm Newton centimeter    -   ETOH ethanol    -   eq. equivalents    -   N normal    -   uPWA variant α-amylase derived from Pyrococcus woesei    -   PWA α-amylase from Pyrococcus woesei    -   MWCO molecular weight cut-off    -   SSRL Stanford Synchrotron Radiation Lightsource    -   PDB Protein Database    -   CAZy Carbohydrate-Active Enzymes database    -   Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride    -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

1.2. Definitions

The terms “amylase” or “amylolytic enzyme” refer to an enzyme that is,among other things, capable of catalyzing the degradation of starch.α-Amylases are hydrolases that cleave the α-D-(1→4) O-glycosidiclinkages in starch. Generally, α-amylases (EC 3.2.1.1; α-D-(1→4)-glucanglucanohydrolase) are defined as endo-acting enzymes cleaving α-D-(1→4)0-glycosidic linkages within the starch molecule in a random fashionyielding polysaccharides containing three or more (1-4)-α-linkedD-glucose units. In contrast, the exo-acting amylolytic enzymes, such asβ-amylases (EC 3.2.1.2; α-D-(1→4)-glucan maltohydrolase) and someproduct-specific amylases like maltogenic α-amylase (EC 3.2.1.133)cleave the polysaccharide molecule from the non-reducing end of thesubstrate. β-amylases, α-glucosidases (EC 3.2.1.20; α-D-glucosideglucohydrolase), glucoamylase (EC 3.2.1.3; α-D-(1→4)-glucanglucohydrolase), and product-specific amylases like themaltotetraosidases (EC 3.2.1.60) and the maltohexaosidases (EC 3.2.1.98)can produce malto-oligosaccharides of a specific length or enrichedsyrups of specific maltooligosaccharides. Some bacterial α-amylasespredominantly produce maltotetraose (G4), maltopentaose (G5) ormaltohexaose (G6) from starch and related α-1,4-glucans, while mostα-amylases further convert them to glucose and or maltose as finalproducts. G6 amylases such as AA560 amylase derived from Bacillus sp.DSM 12649 (i.e., the parent of STAINZYME™) and Bacillus sp. 707 amylase,which are also called maltohexaose-forming α-amylases (EC 3.2.1.98), aretechnically exo acting, but have similar structures compared toα-amylases, and in some cases appear to respond to the some of the samebeneficial mutations.

“Enzyme units” herein refer to the amount of product formed per timeunder the specified conditions of the assay. For example, a“glucoamylase activity unit” (GAU) is defined as the amount of enzymethat produces 1 g of glucose per hour from soluble starch substrate (4%DS) at 60° C., pH 4.2. A “soluble starch unit” (SSU) is the amount ofenzyme that produces 1 mg of glucose per minute from soluble starchsubstrate (4% DS) at pH 4.5, 50° C. DS refers to “dry solids.”

The term “starch” refers to any material comprised of the complexpolysaccharide carbohydrates of plants, comprised of amylose andamylopectin with the formula (C₆H₁₀O₅)_(x), wherein X can be any number.The term includes plant-based materials such as grains, cereal, grasses,tubers and roots, and more specifically materials obtained from wheat,barley, corn, rye, rice, sorghum, brans, cassava, millet, milo, potato,sweet potato, and tapioca. The term “starch” includes granular starch.The term “granular starch” refers to raw, i.e., uncooked starch, e.g.,starch that has not been subject to gelatinization.

The terms, “wild-type,” “parental,” or “reference,” with respect to apolypeptide, refer to a naturally-occurring polypeptide that does notinclude a man-made substitution, insertion, or deletion at one or moreamino acid positions. Similarly, the terms “wild-type,” “parental,” or“reference,” with respect to a polynucleotide, refer to anaturally-occurring polynucleotide that does not include a man-madenucleoside change. However, note that a polynucleotide encoding awild-type, parental, or reference polypeptide is not limited to anaturally-occurring polynucleotide, and encompasses any polynucleotideencoding the wild-type, parental, or reference polypeptide.

Reference to the wild-type polypeptide is understood to include themature form of the polypeptide. A “mature” polypeptide or variant,thereof, is one in which a signal sequence is absent, for example,cleaved from an immature form of the polypeptide during or followingexpression of the polypeptide.

The term “variant,” with respect to a polypeptide, refers to apolypeptide that differs from a specified wild-type, parental, orreference polypeptide in that it includes one or morenaturally-occurring or man-made substitutions, insertions, or deletionsof an amino acid. Similarly, the term “variant,” with respect to apolynucleotide, refers to a polynucleotide that differs in nucleotidesequence from a specified wild-type, parental, or referencepolynucleotide. The identity of the wild-type, parental, or referencepolypeptide or polynucleotide will be apparent from context.

In the case of the present α-amylases, “activity” refers to α-amylaseactivity, which can be measured as described, herein.

The term “recombinant,” when used in reference to a subject cell,nucleic acid, protein or vector, indicates that the subject has beenmodified from its native state. Thus, for example, recombinant cellsexpress genes that are not found within the native (non-recombinant)form of the cell, or express native genes at different levels or underdifferent conditions than found in nature. Recombinant nucleic acidsdiffer from a native sequence by one or more nucleotides and/or areoperably linked to heterologous sequences, e.g., a heterologous promoterin an expression vector. Recombinant proteins may differ from a nativesequence by one or more amino acids and/or are fused with heterologoussequences. A vector comprising a nucleic acid encoding an amylase is arecombinant vector.

“Combinatorial variants” are variants comprising two or more mutations,e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., substitutions, deletions, and/orinsertions.

As used herein, “combinable mutations” are mutations at any amino acidposition that can be used to make combinatorial variants. Combinablemutations improve at least one desired property of the molecule (in thiscase, an α-amylase), while not significantly decreasing eitherexpression, activity, or stability. Combinable mutations can be groupedas follows:

Group A: A mutation that produces a variant wherein the minimumperformance indices (PI) relative to a defined parental protein for: (i)protein expression, (ii) activity, (iii) CS-28 microswatch activity atpH 8 (16° C., 32° C., or 50° C.) or pH10 (16° C. or 50° C.), and (iv)detergent stability or thermostability are greater than or equal to 0.9,and in addition have a PI for any one of these tests that is greaterthan or equal to 1.0.Group B: A mutation that produces a variant wherein the minimumperformance indices (PI) relative to a defined parental protein for: (i)protein expression, (ii) activity, (iii) CS-28 microswatch activity atpH 8 (16° C., 32° C., or 50° C.) or pH10 (16° C. or 50° C.), and (iv)detergent stability or thermostability are greater than or equal to 0.8,and in in addition have a PI for any one of these tests that is greaterthan or equal to 1.2.Group C: A mutation that produces a variant wherein the minimumperformance indices (PI) relative to a defined parental protein for: (i)protein expression, (ii) activity, (iii) CS-28 microswatch activity atpH 8 (16° C., 32° C., or 50° C.) or pH10 (16° C. or 50° C.), and (iv)detergent stability or thermostability are greater than or equal to 0.5,and in addition have a PI for any one of these tests that is greaterthan or equal to 1.5.

The properties of combinable mutations are summarized in the followingTable.

TABLE A Performance properties for each group of combinable mutationsPerformance Index (PI) Synthetic Stability Minimum PI in Cleaningsubstrate (detergent or one or more Group Expression (pH 6 or 8)activity thermal*) tests A ≥0.9 ≥0.9 ≥0.9 ≥0.9 X ≥ 1.0 B ≥0.8 ≥0.8 ≥0.8≥0.8 X ≥ 1.2 C ≥0.5 ≥0.5 ≥0.5 ≥0.5 X ≥ 1.5 *Thermal stability notmeasured for the full SEL libraries

Preferred combinable mutations are at “productive positions,” asdescribed, below. In the case of the present α-amylases, “activity”refers to α-amylase activity, which can be measured as described,herein.

As used herein, “productive positions” are amino acid positions that aretolerant to substitution with different amino acid residues, wherein theresulting variants meet a set of performance criteria for combinability,as set forth above. Productive positions can be assigned a ProductivityScore as follows:

A. For the 24-site SEL libraries: Positions where less than 15% of thesubstitutions at a given position fall within groups A, B, or C aregiven a Productivity Score of “1”. Positions where less than 40%, butgreater than, or equal to 15% of the substitutions at a given positionfall within groups A, B, or C are given a Productivity Score of “2”.Positions where less than 75%, but greater than, or equal to 40% of thesubstitutions at a given position fall within groups A, B, or C aregiven a Productivity Score of “3”. Positions where 75% or more of thesubstitutions at a given position fall within groups A, B, or C aregiven a Productivity Score of “4”.B. For the full SEL libraries: Positions where less than 15% of thesubstitutions at a given position fall within groups A, B, or C aregiven a Productivity Score of “1”. Positions where less than 30%, butgreater than, or equal to 15% of the substitutions at a given positionfall within groups A, B, or C are given a Productivity Score of “2”.Positions where less than 50%, but greater than, or equal to 30% of thesubstitutions at a given position fall within groups A, B, or C aregiven a Productivity Score of “3”. Positions where 50% or more of thesubstitutions at a given position fall within groups A, B, or C aregiven a Productivity Score of “4”.

Preferred productive positions are combinable mutations.

As used herein, “suitability score” refers to the ability of one or morecombinable mutations to be used to make combinatorial variants, based onthe performance criteria for combinability, (i.e., A, B, and C, as setforth, above) in which each of the mutations fall. A higher suitabilityscore indicates a mutation or mutations that are more suitable for usein making combinatorial variants.

Suitability scores are described in the following Table.

TABLE B Definitions of suitability scores Substitutions Occur inGroup(s) Suitability Score A, B and C +++++ A and B ++++ A or (B and C)+++ B ++ C +

The terms “recovered,” “isolated,” and “separated,” refer to a compound,protein (polypeptides), cell, nucleic acid, amino acid, or otherspecified material or component that is removed from at least one othermaterial or component with which it is naturally associated as found innature. An “isolated” polypeptides, thereof, includes, but is notlimited to, a culture broth containing secreted polypeptide expressed ina heterologous host cell.

The term “purified” refers to material (e.g., an isolated polypeptide orpolynucleotide) that is in a relatively pure state, e.g., at least about90% pure, at least about 95% pure, at least about 98% pure, or even atleast about 99% pure.

The term “enriched” refers to material (e.g., an isolated polypeptide orpolynucleotide) that is in about 50% pure, at least about 60% pure, atleast about 70% pure, or even at least about 70% pure.

The terms “thermostable” and “thermostability,” with reference to anenzyme, refer to the ability of the enzyme to retain activity afterexposure to an elevated temperature. The thermostability of an enzyme,such as an amylase enzyme, is measured by its half-life (t_(1/2)) givenin minutes, hours, or days, during which half the enzyme activity islost under defined conditions. The half-life may be calculated bymeasuring residual α-amylase activity following exposure to (i.e.,challenge by) an elevated temperature.

A “pH range,” with reference to an enzyme, refers to the range of pHvalues under which the enzyme exhibits catalytic activity.

The terms “pH stable” and “pH stability,” with reference to an enzyme,relate to the ability of the enzyme to retain activity over a wide rangeof pH values for a predetermined period of time (e.g., 15 min, 30 min.,1 hour).

The term “amino acid sequence” is synonymous with the terms“polypeptide,” “protein,” and “peptide,” and are used interchangeably.Where such amino acid sequences exhibit activity, they may be referredto as an “enzyme.” The conventional one-letter or three-letter codes foramino acid residues are used, with amino acid sequences being presentedin the standard amino-to-carboxy terminal orientation (i.e., N→C).

The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, andsynthetic molecules capable of encoding a polypeptide. Nucleic acids maybe single stranded or double stranded, and may be chemicalmodifications. The terms “nucleic acid” and “polynucleotide” are usedinterchangeably. Because the genetic code is degenerate, more than onecodon may be used to encode a particular amino acid, and the presentcompositions and methods encompass nucleotide sequences that encode aparticular amino acid sequence. Unless otherwise indicated, nucleic acidsequences are presented in 5′-to-3′ orientation.

“Hybridization” refers to the process by which one strand of nucleicacid forms a duplex with, i.e., base pairs with, a complementary strand,as occurs during blot hybridization techniques and PCR techniques.Stringent hybridization conditions are exemplified by hybridizationunder the following conditions: 65° C. and 0.1×SSC (where 1×SSC=0.15 MNaCl, 0.015 M Na₃ citrate, pH 7.0). Hybridized, duplex nucleic acids arecharacterized by a melting temperature (T_(m)), where one-half of thehybridized nucleic acids are unpaired with the complementary strand.Mismatched nucleotides within the duplex lower the T_(m). A nucleic acidencoding a variant α-amylase may have a T_(m) reduced by 1° C.-3° C. ormore compared to a duplex formed between the nucleotide of SEQ ID NO: 2and its identical complement.

A “synthetic” molecule is produced by in vitro chemical or enzymaticsynthesis rather than by an organism.

The terms “transformed,” “stably transformed,” and “transgenic,” usedwith reference to a cell means that the cell contains a non-native(e.g., heterologous) nucleic acid sequence integrated into its genome orcarried as an episome that is maintained through multiple generations.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, “transformation” or“transduction,” as known in the art.

A “host strain” or “host cell” is an organism into which an expressionvector, phage, virus, or other DNA construct, including a polynucleotideencoding a polypeptide of interest (e.g., an amylase) has beenintroduced. Exemplary host strains are microorganism cells (e.g.,bacteria, filamentous fungi, and yeast) capable of expressing thepolypeptide of interest and/or fermenting saccharides. The term “hostcell” includes protoplasts created from cells.

The term “heterologous” with reference to a polynucleotide or proteinrefers to a polynucleotide or protein that does not naturally occur in ahost cell.

The term “endogenous” with reference to a polynucleotide or proteinrefers to a polynucleotide or protein that occurs naturally in the hostcell.

The term “expression” refers to the process by which a polypeptide isproduced based on a nucleic acid sequence. The process includes bothtranscription and translation.

A “selective marker” or “selectable marker” refers to a gene capable ofbeing expressed in a host to facilitate selection of host cells carryingthe gene. Examples of selectable markers include but are not limited toantimicrobials (e.g., hygromycin, bleomycin, or chloramphenicol) and/orgenes that confer a metabolic advantage, such as a nutritional advantageon the host cell.

A “vector” refers to a polynucleotide sequence designed to introducenucleic acids into one or more cell types. Vectors include cloningvectors, expression vectors, shuttle vectors, plasmids, phage particles,cassettes and the like.

An “expression vector” refers to a DNA construct comprising a DNAsequence encoding a polypeptide of interest, which coding sequence isoperably linked to a suitable control sequence capable of effectingexpression of the DNA in a suitable host. Such control sequences mayinclude a promoter to effect transcription, an optional operatorsequence to control transcription, a sequence encoding suitable ribosomebinding sites on the mRNA, enhancers and sequences which controltermination of transcription and translation.

The term “operably linked” means that specified components are in arelationship (including but not limited to juxtaposition) permittingthem to function in an intended manner. For example, a regulatorysequence is operably linked to a coding sequence such that expression ofthe coding sequence is under control of the regulatory sequences.

A “signal sequence” is a sequence of amino acids attached to theN-terminal portion of a protein, which facilitates the secretion of theprotein outside the cell. The mature form of an extracellular proteinlacks the signal sequence, which is cleaved off during the secretionprocess.

“Biologically active” refer to a sequence having a specified biologicalactivity, such an enzymatic activity.

The term “specific activity” refers to the number of moles of substratethat can be converted to product by an enzyme or enzyme preparation perunit time under specific conditions. Specific activity is generallyexpressed as units (U)/mg of protein.

As used herein, “water hardness” is a measure of the minerals (e.g.,calcium and magnesium) present in water.

A “swatch” is a piece of material such as a fabric that has a stainapplied thereto. The material can be, for example, fabrics made ofcotton, polyester or mixtures of natural and synthetic fibers. Theswatch can further be paper, such as filter paper or nitrocellulose, ora piece of a hard material such as ceramic, metal, or glass. Foramylases, the stain is starch based, but can include blood, milk, ink,grass, tea, wine, spinach, gravy, chocolate, egg, cheese, clay, pigment,oil, or mixtures of these compounds.

A “smaller swatch” is a section of the swatch that has been cut with asingle hole punch device, or has been cut with a custom manufactured96-hole punch device, where the pattern of the multi-hole punch ismatched to standard 96-well microtiter plates, or the section has beenotherwise removed from the swatch. The swatch can be of textile, paper,metal, or other suitable material. The smaller swatch can have the stainaffixed either before or after it is placed into the well of a 24-, 48-or 96-well microtiter plate. The smaller swatch can also be made byapplying a stain to a small piece of material. For example, the smallerswatch can be a stained piece of fabric ⅝″ or 0.25″ in diameter. Thecustom manufactured punch is designed in such a manner that it delivers96 swatches simultaneously to all wells of a 96-well plate. The deviceallows delivery of more than one swatch per well by simply loading thesame 96-well plate multiple times. Multi-hole punch devices can beconceived of to deliver simultaneously swatches to any format plate,including but not limited to 24-well, 48-well, and 96-well plates. Inanother conceivable method, the soiled test platform can be a bead madeof metal, plastic, glass, ceramic, or another suitable material that iscoated with the soil substrate. The one or more coated beads are thenplaced into wells of 96-, 48-, or 24-well plates or larger formats,containing suitable buffer and enzyme.

“A cultured cell material comprising an amylase” or similar language,refers to a cell lysate or supernatant (including media) that includesan amylase as a component. The cell material may be from a heterologoushost that is grown in culture for the purpose of producing the amylase.

“Percent sequence identity” means that a particular sequence has atleast a certain percentage of amino acid residues identical to those ina specified reference sequence, when aligned using the CLUSTAL Walgorithm with default parameters. See Thompson et al. (1994) NucleicAcids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithmare:

-   -   Gap opening penalty: 10.0    -   Gap extension penalty: 0.05    -   Protein weight matrix: BLOSUM series    -   DNA weight matrix: IUB    -   Delay divergent sequences %: 40    -   Gap separation distance: 8    -   DNA transitions weight: 0.50    -   List hydrophilic residues: GPSNDQEKR    -   Use negative matrix: OFF    -   Toggle Residue specific penalties: ON    -   Toggle hydrophilic penalties: ON    -   Toggle end gap separation penalty OFF.

Deletions are counted as non-identical residues, compared to a referencesequence. Deletions occurring at either termini are included. Forexample, a variant with five amino acid deletions of the C-terminus ofthe mature CspAmy2 polypeptide of SEQ ID NO: 1 would have a percentsequence identity of 99% (612/617 identical residues×100, rounded to thenearest whole number) relative to the mature polypeptide. Such a variantwould be encompassed by a variant having “at least 99% sequenceidentity” to a mature amylase polypeptide.

“Fused” polypeptide sequences are connected, i.e., operably linked, viaa peptide bond between two subject polypeptide sequences.

The term “filamentous fungi” refers to all filamentous forms of thesubdivision Eumycotina, particularly Pezizomycotina species.

The term “degree of polymerization” (DP) refers to the number (n) ofanhydro-glucopyranose units in a given saccharide. Examples of DP1 arethe monosaccharides glucose and fructose. Examples of DP2 are thedisaccharides maltose and sucrose. The term “DE,” or “dextroseequivalent,” is defined as the percentage of reducing sugar, i.e.,D-glucose, as a fraction of total carbohydrate in a syrup.

The term “dry solids content” (ds) refers to the total solids of aslurry in a dry weight percent basis. The term “slurry” refers to anaqueous mixture containing insoluble solids.

The phrase “simultaneous saccharification and fermentation (SSF)” refersto a process in the production of biochemicals in which a microbialorganism, such as an ethanologenic microorganism, and at least oneenzyme, such as an amylase, are present during the same process step.SSF includes the contemporaneous hydrolysis of starch substrates(granular, liquefied, or solubilized) to saccharides, including glucose,and the fermentation of the saccharides into alcohol or otherbiochemical or biomaterial in the same reactor vessel.

An “ethanologenic microorganism” refers to a microorganism with theability to convert a sugar or oligosaccharide to ethanol.

The term “fermented beverage” refers to any beverage produced by amethod comprising a fermentation process, such as a microbialfermentation, e.g., a bacterial and/or fungal fermentation. “Beer” is anexample of such a fermented beverage, and the term “beer” is meant tocomprise any fermented wort produced by fermentation/brewing of astarch-containing plant material. Often, beer is produced exclusivelyfrom malt or adjunct, or any combination of malt and adjunct. Examplesof beers include: full malted beer, beer brewed under the“Reinheitsgebot,” ale, India pale ale, lager, pilsner, bitter, Happoshu(second beer), third beer, dry beer, near beer, light beer, low alcoholbeer, low calorie beer, porter, bock, dopplebock, stout, porter, maltliquor, non-alcoholic beer, non-alcoholic malt liquor and the like, butalso alternative cereal and malt beverages such as fruit flavored maltbeverages, e.g., citrus flavored, such as lemon-, orange-, lime-, orberry-flavored malt beverages, liquor flavored malt beverages, e.g.,vodka-, rum-, or tequila-flavored malt liquor, or coffee flavored maltbeverages, such as caffeine-flavored malt liquor, and the like.

The term “malt” refers to any malted cereal grain, such as malted barleyor wheat.

The term “adjunct” refers to any starch and/or sugar containing plantmaterial that is not malt, such as barley or wheat malt. Examples ofadjuncts include common corn grits, refined corn grits, brewer's milledyeast, rice, sorghum, refined corn starch, barley, barley starch,dehusked barley, wheat, wheat starch, torrified cereal, cereal flakes,rye, oats, potato, tapioca, cassava and syrups, such as corn syrup,sugar cane syrup, inverted sugar syrup, barley and/or wheat syrups, andthe like.

The term “mash” refers to an aqueous slurry of any starch and/or sugarcontaining plant material, such as grist, e.g., comprising crushedbarley malt, crushed barley, and/or other adjunct or a combinationthereof, mixed with water later to be separated into wort and spentgrains.

The term “wort” refers to the unfermented liquor run-off followingextracting the grist during mashing.

“Iodine-positive starch” or “IPS” refers to (1) amylose that is nothydrolyzed after liquefaction and saccharification, or (2) a retrogradedstarch polymer. When saccharified starch or saccharide liquor is testedwith iodine, the high DPn amylose or the retrograded starch polymerbinds iodine and produces a characteristic blue color. The saccharideliquor is thus termed “iodine-positive saccharide,” “blue saccharide,”or “blue sac.”

The terms “retrograded starch” or “starch retrogradation” refer tochanges that occur spontaneously in a starch paste or gel on ageing.

The term “about” refers to ±15% to the referenced value.

2. α-Amylase Variants

An aspect of the present compositions and methods is variant amylaseenzymes discovered using a combination of experimental approaches,including the use of site evaluation libraries (SELs) andstructure-based analysis.

2.1 α-Amylase Variants Based on SEL Libraries of Amy707 α-Amylase

In one aspect, variant α-amylase polypeptides are provided. The variantamylases have one or more mutations, as set forth, herein, with respectto a parental α-amylase having a similar fold and/or 60% or greateramino acid sequence identity to Bacillus sp. 707 amylase (SEQ ID NO: 3)or AA560 amylase (SEQ ID NO: 4).

In some embodiments, the parent enzyme is Amy707 α-amylase derived fromBacillus sp.707 (#707) having the amino acid sequence of SEQ ID NO: 3:

HHNGTNGTMM QYFEWYLPND GNHWNRLNSD ASNLKSKGIT AVWIPPAWKG ASQNDVGYGAYDLYDLGEFN QKGTVRTKYG TRSQLQAAVT SLKNNGIQVY GDVVMNHKGG ADATEMVRAVEVNPNNRNQE VTGEYTIEAW TRFDFPGRGN THSSFKWRWY HFDGVDWDQS RRLNNRIYKFRGHGKAWDWE VDTENGNYDY LMYADIDMDH PEVVNELRNW GVWYTNTLGL DGFRIDAVKHIKYSFTRDWI NHVRSATGKN MFAVAEFWKN DLGAIENYLQ KTNWNHSVFD VPLHYNLYNASKSGGNYDMR NIFNGTVVQR HPSHAVTFVD NHDSQPEEAL ESFVEEWFKP LAYALTLTREQGYPSVFYGD YYGIPTHGVP AMRSKIDPIL EARQKYAYGK QNDYLDHHNI IGWTREGNTAHPNSGLATIM SDGAGGSKWM FVGRNKAGQV WSDITGNRTG TVTINADGWG NFSVNGGSVS IWVNK

In some embodiments, the parent enzyme is AA560 α-amylase derived fromBacillus sp. DSM 12649 having the amino acid sequence of SEQ ID NO: 4:

HHNGTNGTMM QYFEWYLPND GNHWNRLRSD ASNLKDKGIS AVWIPPAWKG ASQNDVGYGAYDLYDLGEFN QKGTIRTKYG TRNQLQAAVN ALKSNGIQVY GDVVMNHKGG ADATEMVRAVEVNPNNRNQE VSGEYTIEAW TKFDFPGRGN THSNFKWRWY HFDGVDWDQS RKLNNRIYKFRGDGKGWDWE VDTENGNYDY LMYADIDMDH PEVVNELRNW GVWYTNTLGL DGFRIDAVKHIKYSFTRDWI NHVRSATGKN MFAVAEFWKN DLGAIENYLN KTNWNHSVFD VPLHYNLYNASKSGGNYDMR QIFNGTVVQR HPMHAVTFVD NHDSQPEEAL ESFVEEWFKP LAYALTLTREQGYPSVFYGD YYGIPTHGVP AMKSKIDPIL EARQKYAYGR QNDYLDHHNI IGWTREGNTAHPNSGLATIM SDGAGGNKWM FVGRNKAGQV WTDITGNRAG TVTINADGWG NFSVNGGSVS IWVNK

α-amylase variants that include combinable mutations were identified bymaking a site evaluation library (SEL) based on Amy707 (SEQ ID NO: 3)and testing the resulting variants for various performance criteria,such as detergent stability, thermostability, cleaning performance, andexpression levels, the detailed procedures for which are described inthe Examples or otherwise known. Each variant was assayed for thedifferent enzymatic and biochemical properties, and characterized by aperformance index (PI) value, which compared the relative performance ofthe variant to Amy707 amylase for each performance criteria. A PI thatis greater than 1 (i.e., PI>1) indicated improved performance by avariant as compared to Amy707, while a PI of 1 (i.e., PI=1) indicated avariant that performed the same as the Amy707, and a PI that is lessthan 1 (i.e., PI<1) indicated a variant that performed worse than theAmy707. PI values were then used to identify combinable mutations andproductive positions.

Combinable mutations are mutations at any amino acid position thatimprove at least one desired property of the molecule, while notsignificantly decreasing expression, activity, or stability. Combinablemutations are assigned to one of three Groups (i.e., A, B, or C), as setforth, herein. Preferred combinable mutations are at productivepositions. Productive positions are amino acid positions that aretolerant to substitution with different amino acid residues, wherein theresulting variants meet a set of performance criteria for combinability,as set forth herein.

Combinable mutations and productive positions are not to be confusedwith previously-identified, single-site mutations, some of which havesubsequently been found by trial and error to work in combination withother mutations. Previously-identified, single-site mutations areinvariably “winners” with respect to improving any one performance orstability feature. While this makes them attractive mutations to includein varient amylases, these “winners” tend to adversly affect otherperformance or stability features of the variants, which often requiresmaking additional mutations to correct the defects.

In contrast, combinable mutations may be only incrementally beneficialin improving any one performance or stability feature of an variantamylase. However, they are carefully selected to be minimallydetrimental to other desired performance or stability features, makingthem well suited for use in combination with other combinable mutationsto contruct variant amylases having desired improved enzymatic andbiochemical properties without being crippled in others, resulting inrobust variants having a good balance of performance, stability, andexpression potential.

Further based on measured enzymatic and biochemical properties of thevariant amylases, the suitability scores of the different mutations formaking combinatorial variants were determined. The suitabilty scorerefers to the ability of one or more combinable mutations to be used tomake combinatorial variants, based on the performance criteria forcombinability (i.e., A, B, and C, as set forth, above), in which each ofthe mutations fall.

The suitability scores of individual substitutions in Amy707 are shownin Table C. The position numbering is based on the amino acid sequenceof the mature Amy707 polypeptide (SEQ ID NO: 3). Wild-type residues atthe indicated positions are given a suitability score of +++.Substitutions more likely to be combinable with other mutations aregiven a suitability score of ++++, or even +++++. In general, preferredsuitabilty scores are +++, ++++, or +++++, ++++ or +++++, or even +++++.

TABLE C Suitability scores of individual substitutions in Amy707VARIANTS SUITABILITY SCORE POS Prod. Score (+) (++) (+++)* (++++)(+++++) 1 4 E C HFKNQRT AILMW 2 4 A HCDEFGIKNPQ LM SW 3 4 NCDFKLQSTV AEM4 4 DKM GFHPSTW EIL 5 4 CN THIQSVW DGM A 6 3 EGQT NS A 7 4 A DILTY GMSHPQRV 10 1 IL M 12 1 Y A 16 3 HT YAENW D 17 3 D V LS AGT 18 1 AE P 19 1L N D 20 3 EHI DS GNY AC 22 4 GMV I NELQSTW R 23 2 HQ FMT 25 4 M NS TVACGKY 26 2 RKQT 27 2 A LIV 28 4 NADQWY CEGHKR 29 4 SCDEFHKMRTV AN WY 302 DEMNQR 31 1 AS 32 4 CDEG SN MWY ILQR 33 4 NHIKQTVWY CDMR 34 1 LFM 35 4CEFIL KMNQ AGH 36 2 SDGKQT 40 1 K T N 41 3 IKM DQ A S C 47 2 GMP AS 50 2GC S 52 2 L ST KM R 53 1 Q A 54 4 M NADEFGQSVW C 56 2 NS V E 61 1 Y F 632 NQ LM 64 1 YH 66 1 V L M 68 2 D EA Q 70 4 R NEGHKV CDFIMS L 72 1 K R73 4 D EW GQRT KMSY 74 2 G T S 75 1 M VI 77 2 A S TI NV 81 3 TACDFIKNPS82 3 Q RACFSVY IKM 83 2 M SKNQRT 84 3 CFL QEN DK M 86 4 H QEIRTVWY K 873 ADKT M 88 1 A M 89 2 VAC I 90 2 TGMQRS 91 3 M SHKQRTV AEN 93 1 H KR 944 NFH ACDGKLM QR 95 4 G D NCFHIQRSTY A 96 2 DEN G 97 1 V I 98 3 QCDEGHKRA 99 2 A VC I 100 2 Y CFI 101 1 A G 103 2 F I VL CT A 110 2 PS GA 111 1S A 112 1 C D E 113 4 IVY A CEFGHKMR 115 1 E Q 116 4 PV MDIQ ACEFGLNRW T117 3 L VEPS R T 118 3 DLV RW EQT G 119 1 AC S 122 1 VC 123 2 ACL N 1241 NT P 125 4 Y NGHISTW CFLMR 126 1 N D 128 2 E N LY C 129 1 V Q 132 1 TS133 3 GAHQST DP 134 2 V ES DT P 135 2 Q YFL CM 136 4 FY TCKLQR DGMNP 1382 DLMN E 139 2 CG A 140 1 WF Y 142 3 T RS CEFGHKY 144 3 DEISY KM 145 1FMY 146 4 ACDEFGH P MRSWY 147 2 GDIL A 149 4 PW GCDEFHKRV AL 150 2 L HMNPS R 151 4 D TEGHILMQV 153 1 N S 154 2 LRY S 155 1 W F 156 2 AD KS 1582 AL RKQ CN 160 4 GILMP F YAC HQS DEKNR 162 1 M F 165 1 V CT 167 1 M F W168 1 C D 169 4 FGIKWY S QCMN ADEHV 170 2 SC AEK 171 2 RT MS 172 4 YRCEGHQ AMS 173 4 DKN L ACFHWY 174 4 NDGHILPSTV 175 3 M NRS ADEL 176 1 KTR 178 1 W Y 179 2 CL KM Q 181 4 ALNT REF CIMQV SY 182 1 C GD 183 4 NHCQVWY DEFLMP A 184 2 AEL D G N C 186 2 AG EN D 195 2 FY NW L 196 1 G C203 1 Y N 206 2 NS H IM C T 210 3 ACEMNQRS H D 211 4 L P CDMNQS AFKV 2153 FL NKM DQ 216 3 GLN EY ACST HQ 217 1 M L 218 3 ACEFHM K R 219 3 ACMNRT D 221 1 IV G 222 4 EM VDFGNY AHILRST 225 2 A TR K 226 2 Y NDE AK 2272 TE AK 228 1 L IM 229 2 FV C G 230 1 M L 231 2 DE GT 233 3 L CHMY FAW235 2 ILMV 238 1 I V 243 1 YF 244 2 S N DEHQ 245 1 E M F 247 2 L RT AE249 2 M FL W 250 2 ILMV 251 4 AH NPTVY CGW KLQRS 252 2 HDEKN 253 1 M V257 2 TMS A 258 3 IMV PS GDN K R 259 3 DERT KCP AGHQ 260 2 N DP KR 261 3G MCI AEQT 262 4 ACDEGLQS HKRY FM 263 1 AS 265 1 AS G 273 4 CK GVDEHLMPQSTY 276 1 C ET 280 2 MV QDHK N 283 1 NG D 285 2 NEKST M 286 3 F NHLV ACEM T 287 2 ADNY S 288 2 L C V IT 292 1 LM P 296 1 NQ 297 1 L M 2982 YFRW 299 3 MY NGHRST 301 1 SAG 302 2 T KCM EQS R 303 4 SEQR ACDGLM 3042 C GKRSV 306 2 NG AD 307 1 YAF 310 1 RQS 311 4 NFT DEGHKLMQRY 312 2 LIV M 313 1 MY F 314 4 M NADEGHIKLST Q VY 317 1 L V 318 2 MS VCI LT 319 3Y QADEGHNR 320 4 DY RHNST AEKMQ 321 1 HWY 322 1 D P 323 4 A P SFHILCGRVY DEMT 324 2 ACM K H Y 326 3 H C VNT AM 327 1 T L 328 1 FV 329 1 V I334 1 T S 337 3 Y A ECDST NQ 339 2 GT A S 341 2 FGK EH ADY 343 1 FTY 3441 VI C 345 4 EAGHKLMNQS TY 346 4 E ACHKRTVY DGMNQS 347 1 A D W 350 1 PE351 2 AC LM Q 352 1 AS 354 1 AS 355 2 LIKMV 356 2 Q CV T IL 357 2 H A LM 358 2 AI G T C 359 2 I W RV 360 4 EACFHLNPQRT K VY 361 4 V HT QDSW CAEG 363 3 IW V YE ADKNQ M 364 2 CG P A 365 2 GV S AN 366 2 C V IL 367 1FY 368 2 GL Y MQ 369 1 S A G 372 3 R YHIKM QTV 374 2 C IQ NS 375 4 QPDGHIRTVY AEKM 377 2 A HKM GT 378 2 L GEHMN 379 3 AL VIQS MNRY 380 2PDEGHKQS 381 2 AGNQRST 382 1 K M 386 1 LV I 387 2 EN DG 388 4 HNSPACFGKLQRTVY D 389 3 F IV EGLMQS 390 1 M LV 391 4 S ECHIW AGKLNR 392 2ACG S 394 3 EH QCDGLRV Y 395 4 V KDEMST AGQR 396 2 YN K M 397 1 AG 400 4AI KF GHLMQTVW 401 1 QH M 402 3 V NCIKLSY 403 1 ET D 405 2 A LCMNTV 4062 ACL Q D N 408 4 HEGNQRST KMP 410 1 N I 411 1 I V 412 1 GAS 413 2 WFHIYL 414 2 TACV S 415 2 W CY R 416 4 EFHQRTVWY ADGKLN 417 1 A G 418 4 INADKMQSTV L 419 4 TDEHKLNPQRW MSY 420 4 DFGHILM A QRSTVW 421 4 V HCIDEKLMRWY AN 422 4 A PGVY CEFLMT 423 4 NCDEFHILRST 424 4 N EV SCDG AIQTW425 1 GA 426 2 L ANS 427 1 ACT 428 1 TNS 429 1 IM 430 2 MGIL V 431 1 ACS 433 3 M C GDEKNR A 434 4 ACDEFHIKNPQ M RSTV 435 4 ET GKMQR ACNP 436 2ACQ D GS 437 3 C SKNT AD 438 2 CE KS H 439 2 L WH MQ 441 2 FH NY 442 1 AVC 444 2 AQ RK 445 3 C NGKRT AEQ 446 3 T Y KCFS AHMQ 448 1 F N G 450 3AL VEIQRST 451 1 WF 452 4 H SCEFQTW AKNY 454 3 F CMS IAV L 457 2 T NGHQR458 4 L D RHSTVY CEKMN 459 4 ADEGH CP TL NS 460 3 KN GQ EHS 461 4 F TDVACEGKLNPQRY 463 2 L TEKPQR 465 2 Q N DG 466 4 ADEGKNPQRS 467 1 D E 469 1Y W 471 3 NHQRY CDE 473 1 SP 474 1 V S 475 1 NDE 476 2 HN GDR E 477 3 PTDK GAR NQ 478 1 A SG 479 1 VT 481 2 I LTV 482 1 WY 483 3 VHMR CGST 484 3NAEGHQRS 485 3 MP KHQST *The first listed amino acid residue is thewild-type residue.

The suitability scores of a subset of substitutions in Amy707, whichwere identified in limted, 24-site SEL libraries, are shown in Table D.As before, position numbering is based on the amino acid sequence of themature Amy707 polypeptide (SEQ ID NO: 3), wild-type residues at theindicated positions are given a suitability score of +++, substitutionsmore likely to be combinable with other mutations are given asuitability score of ++++, or even +++++, and, in general, preferredsuitabilty scores are +++, ++++, or +++++, ++++ or +++++, or even +++++.

TABLE D Suitability scores of a subset of individual substitutions inAmy707 Pro- duc- tivity VARIANTS SUITABILITY SCORE POS score (+) (++)(+++)* (++++) (+++++) 1 3 HILTFWQ ACK RM 83 3 A SCTNG IRK 125 4 RTYNLMVGH ISFW C 128 2 EY D NL C 131 2 RKS VCT 160 4 L YIG ARKSHQ CDEN 1793 LEVNW KICM Q G 183 4 T HGLF RCSDEMVNW AP YQ 184 3 I ALQ G CN DE 186 2R ADSG EMN 244 2 STN KDEHQ 280 2 CT QDE IKN 306 3 NIKDTEVG AR 320 3 HQRDEN AST K 321 2 MV HFY 380 3 PDTEGHQ C KS 408 3 HIMNPQ RSTEG K 434 4 LAIRDEMGPHQ CKSTVN 454 2 M ICSV 475 2 NRCSD 476 2 GRNHQ CDE 477 2 T AGKDNQ R 484 3 W DG NTQ ARS *The first listed amino acid residue is thewild-type residue.

While evaluating mutations based on suitabilility score represents onerefined aspect of the present compositions and methods, theidentification of productive positions, which are tolerant tosubstitution with different amino acid residues, represents a number ofsignificant embodiments.

Each productive position identified in the following lists withspecified criteria, and each substitution identified in parenthesisfollowing the numerical position identifier in each of these lists,represents a mutation, identified by experimental data, that eitherdirectly contributes to the performance of an amylase variant, or isdetermined to be combinable with other mutations to produce a amylasevariant with improved performance.

The productive positions in Amy 707 that fall within the previouslydescribed Productivity Scores of “4” (for the full SEL libraries) andthe substitutions within those positions that are combinable are listed,below, in LIST A. Position numbering is based on the mature Amy707protein listed in SEQ ID NO: 3.

List A

1(H, A, C, E, F, I, K, L, M, N, Q, R, T, W); 2(H, A, C, D, E, F, G, I,K, L, M, N, P, Q, S, W); 3(N, A, C, D, E, F, K, L, M, Q, S, T, V); 4(G,D, E, F, H, I, K, L, M, P, S, T, W); 5(T, A, C, D, G, H, I, M, N, Q, S,V, W); 7(G, A, D, H, I, L, M, P, Q, R, S, T, V, Y); 22(N, E, G, I, L, M,Q, R, S, T, V, W); 25(N, A, C, G, K, M, S, T, V, Y); 28(N, A, C, D, E,G, H, K, Q, R, W, Y); 29(S, A, C, D, E, F, H, K, M, N, R, T, V, W, Y);32(S, C, D, E, G, I, L, M, N, Q, R, W, Y); 33(N, C, D, H, I, K, M, Q, R,T, V, W, Y); 35(K, A, C, E, F, G, H, I, L, M, N, Q); 54(N, A, C, D, E,F, G, M, Q, S, V, W); 70(N, C, D, E, F, G, H, I, K, L, M, R, S, V);73(G, D, E, K, M, Q, R, S, T, W, Y); 86(Q, E, H, I, K, R, T, V, W, Y);94(N, A, C, D, F, G, H, K, L, M, Q, R); 95(N, A, C, D, F, G, H, I, Q, R,S, T, Y); 113(A, C, E, F, G, H, I, K, M, R, V, Y); 116(M, A, C, D, E, F,G, I, L, N, P, Q, R, T, V, W); 125(N, C, F, G, H, I, L, M, R, S, T, W,Y); 136(T, C, D, F, G, K, L, M, N, P, Q, R, Y); 146(P, A, C, D, E, F, G,H, M, R, S, W, Y); 149(G, A, C, D, E, F, H, K, L, P, R, V, W); 151(T, D,E, G, H, I, L, M, Q, V); 160(Y, A, C, D, E, F, G, H, I, K, L, M, N, P,Q, R, S); 169(Q, A, C, D, E, F, G, H, I, K, M, N, S, V, W, Y); 172(R, A,C, E, G, H, M, Q, S, Y); 173(L, A, C, D, F, H, K, N, W, Y); 174(N, D, G,H, I, L, P, S, T, V); 181(R, A, C, E, F, I, L, M, N, Q, S, T, V, Y);183(H, A, C, D, E, F, L, M, N, P, Q, V, W, Y); 211(P, A, C, D, F, K, L,M, N, Q, S, V); 222(V, A, D, E, F, G, H, I, L, M, N, R, S, T, Y); 251(N, A, C, G, H, K, L, P, Q, R, S, T, V, W, Y); 262(F, A, C, D, E, G, H,K, L, M, Q, R, S, Y); 273(G, C, D, E, H, K, L, M, P, Q, S, T, V, Y);303(S, A, C, D, E, G, L, M, Q, R); 311(N, D, E, F, G, H, K, L, M, Q, R,T, Y); 314(N, A, D, E, G, H, I, K, L, M, Q, S, T, V, Y); 320(R, A, D, E,H, K, M, N, Q, S, T, Y); 323(S, A, C, D, E, F, G, H, I, L, M, P, R, T,V, Y); 345(E, A, G, H, K, L, M, N, Q, S, T, Y); 346(E, A, C, D, G, H, K,M, N, Q, R, S, T, V, Y); 360(E, A, C, F, H, K, L, N, P, Q, R, T, V, Y);361(Q, A, C, D, E, G, H, S, T, V, W); 375(P, A, D, E, G, H, I, K, M, Q,R, T, V, Y); 388(P, A, C, D, F, G, H, K, L, N, Q, R, S, T, V, Y); 391(E,A, C, G, H, I, K, L, N, R, S, W); 395(K, A, D, E, G, M, Q, R, S, T, V);400(K, A, F, G, H, I, L, M, Q, T, V, W); 408(H, E, G, K, M, N, P, Q, R,S, T); 416(E, A, D, F, G, H, K, L, N, Q, R, T, V, W, Y); 418(N, A, D, I,K, L, M, Q, S, T, V); 419(T, D, E, H, K, L, M, N, P, Q, R, S, W, Y);420(A, D, F, G, H, I, L, M, Q, R, S, T, V, W); 421(H, A, C, D, E, I, K,L, M, N, R, V, W, Y); 422(P, A, C, E, F, G, L, M, T, V, Y); 423(N, C, D,E, F, H, I, L, R, S, T); 424(S, A, C, D, E, G, I, N, Q, T, V, W); 434(A,C, D, E, F, H, I, K, M, N, P, Q, R, S, T, V); 435(G, A, C, E, K, M, N,P, Q, R, T); 452(S, A, C, E, F, H, K, N, Q, T, W, Y); 458(R, C, D, E, H,K, L, M, N, S, T, V, Y); 459(T, A, C, D, E, G, H, L, N, P, S); 461(T, A,C, D, E, F, G, K, L, N, P, Q, R, V, Y); and 466(A, D, E, G, K, N, P, Q,R, S).

The productive positions in Amy 707 that fall within the previouslydescribed Productivity Scores of “3 and 4” (for the full SEL libraries)and the substitutions within those positions that are combinable arelisted, below, in LIST B. Position numbering is based on the matureAmy707 protein listed in SEQ ID NO: 3.

List B

1(H, A, C, E, F, I, K, L, M, N, Q, R, T, W); 2(H, A, C, D, E, F, G, I,K, L, M, N, P, Q, S, W); 3(N, A, C, D, E, F, K, L, M, Q, S, T, V); 4(G,D, E, F, H, I, K, L, M, P, S, T, W); 5(T, A, C, D, G, H, I, M, N, Q, S,V, W); 6(N, A, E, G, Q, S, T); 7(G, A, D, H, I, L, M, P, Q, R, S, T, V,Y); 16(Y, A, D, E, H, N, T, W); 17(L, A, D, G, S, T, V); 20(D, A, C, E,G, H, I, N, S, Y); 22(N, E, G, I, L, M, Q, R, S, T, V, W); 25(N, A, C,G, K, M, S, T, V, Y); 28(N, A, C, D, E, G, H, K, Q, R, W, Y); 29(S, A,C, D, E, F, H, K, M, N, R, T, V, W, Y); 32(S, C, D, E, G, I, L, M, N, Q,R, W, Y); 33(N, C, D, H, I, K, M, Q, R, T, V, W, Y); 35(K, A, C, E, F,G, H, I, L, M, N, Q); 41(A, C, D, I, K, M, Q, S); 54(N, A, C, D, E, F,G, M, Q, S, V, W); 70(N, C, D, E, F, G, H, I, K, L, M, R, S, V); 73(G,D, E, K, M, Q, R, S, T, W, Y); 81(T, A, C, D, F, I, K, N, P, S); 82(R,A, C, F, I, K, M, Q, S, V, Y); 84(Q, C, D, E, F, K, L, M, N); 86(Q, E,H, I, K, R, T, V, W, Y); 87(A, D, K, M, T); 91(S, A, E, H, K, M, N, Q,R, T, V); 94(N, A, C, D, F, G, H, K, L, M, Q, R); 95(N, A, C, D, F, G,H, I, Q, R, S, T, Y); 98(Q, A, C, D, E, G, H, K, R); 113(A, C, E, F, G,H, I, K, M, R, V, Y); 116(M, A, C, D, E, F, G, I, L, N, P, Q, R, T, V,W); 117(V, E, L, P, R, S, T); 118(R, D, E, G, L, Q, T, V, W); 125(N, C,F, G, H, I, L, M, R, S, T, W, Y); 133(G, A, D, H, P, Q, S, T); 136(T, C,D, F, G, K, L, M, N, P, Q, R, Y); 142(R, C, E, F, G, H, K, S, T, Y);144(D, E, I, K, M, S, Y); 146(P, A, C, D, E, F, G, H, M, R, S, W, Y);149(G, A, C, D, E, F, H, K, L, P, R, V, W); 151(T, D, E, G, H, I, L, M,Q, V); 160(Y, A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S); 169(Q, A,C, D, E, F, G, H, I, K, M, N, S, V, W, Y); 172(R, A, C, E, G, H, M, Q,S, Y); 173(L, A, C, D, F, H, K, N, W, Y); 174(N, D, G, H, I, L, P, S, T,V); 175(N, A, D, E, L, M, R, S); 181(R, A, C, E, F, I, L, M, N, Q, S, T,V, Y); 183(H, A, C, D, E, F, L, M, N, P, Q, V, W, Y); 210(H, A, C, D, E,M, N, Q, R, S); 211(P, A, C, D, F, K, L, M, N, Q, S, V); 215(N, D, F, K,L, M, Q); 216(E, A, C, G, H, L, N, Q, S, T, Y); 218(R, A, C, E, F, H, K,M); 219(N, A, C, D, M, R, T); 222(V, A, D, E, F, G, H, I, L, M, N, R, S,T, Y); 233(F, A, C, H, L, M, W, Y); 251(N, A, C, G, H, K, L, P, Q, R, S,T, V, W, Y); 258(G, D, I, K, M, N, P, R, S, V); 259(K, A, C, D, E, G, H,P, Q, R, T); 261(M, A, C, E, G, I, Q, T); 262(F, A, C, D, E, G, H, K, L,M, Q, R, S, Y); 273(G, C, D, E, H, K, L, M, P, Q, S, T, V, Y); 286(H, A,C, E, F, L, M, N, T, V); 299(N, G, H, M, R, S, T, Y); 303(S, A, C, D, E,G, L, M, Q, R); 311(N, D, E, F, G, H, K, L, M, Q, R, T, Y); 314(N, A, D,E, G, H, I, K, L, M, Q, S, T, V, Y); 319(Q, A, D, E, G, H, N, R, Y);320(R, A, D, E, H, K, M, N, Q, S, T, Y); 323(S, A, C, D, E, F, G, H, I,L, M, P, R, T, V, Y); 326(V, A, C, H, M, N, T); 337(E, A, C, D, N, Q, S,T, Y); 345(E, A, G, H, K, L, M, N, Q, S, T, Y); 346(E, A, C, D, G, H, K,M, N, Q, R, S, T, V, Y); 360(E, A, C, F, H, K, L, N, P, Q, R, T, V, Y);361(Q, A, C, D, E, G, H, S, T, V, W); 363(Y, A, D, E, I, K, M, N, Q, V,W); 372(Y, H, I, K, M, Q, R, T, V); 375(P, A, D, E, G, H, I, K, M, Q, R,T, V, Y); 379(V, A, I, L, M, N, Q, R, S, Y); 388(P, A, C, D, F, G, H, K,L, N, Q, R, S, T, V, Y); 389(I, E, F, G, L, M, Q, S, V); 391(E, A, C, G,H, I, K, L, N, R, S, W); 394(Q, C, D, E, G, H, L, R, V, Y); 395(K, A, D,E, G, M, Q, R, S, T, V); 400(K, A, F, G, H, I, L, M, Q, T, V, W); 402(N,C, I, K, L, S, V, Y); 408(H, E, G, K, M, N, P, Q, R, S, T); 416(E, A, D,F, G, H, K, L, N, Q, R, T, V, W, Y); 418(N, A, D, I, K, L, M, Q, S, T,V); 419(T, D, E, H, K, L, M, N, P, Q, R, S, W, Y); 420(A, D, F, G, H, I,L, M, Q, R, S, T, V, W); 421(H, A, C, D, E, I, K, L, M, N, R, V, W, Y);422(P, A, C, E, F, G, L, M, T, V, Y); 423(N, C, D, E, F, H, I, L, R, S,T); 424(S, A, C, D, E, G, I, N, Q, T, V, W); 433(G, A, C, D, E, K, M, N,R); 434(A, C, D, E, F, H, I, K, M, N, P, Q, R, S, T, V); 435(G, A, C, E,K, M, N, P, Q, R, T); 437(S, A, C, D, K, N, T); 445(N, A, C, E, G, K, Q,R, T); 446(K, A, C, F, H, M, Q, S, T, Y); 450(V, A, E, I, L, Q, R, S,T); 452(S, A, C, E, F, H, K, N, Q, T, W, Y); 454(I, A, C, F, L, M, S,V); 458(R, C, D, E, H, K, L, M, N, S, T, V, Y); 459(T, A, C, D, E, G, H,L, N, P, S); 460(G, E, H, K, N, Q, S); 461(T, A, C, D, E, F, G, K, L, N,P, Q, R, V, Y); 466(A, D, E, G, K, N, P, Q, R, S); 471(N, C, D, E, H, Q,R, Y); 477(G, A, D, K, N, P, Q, R, T); 483(V, C, G, H, M, R, S, T);484(N, A, E, G, H, Q, R, S); and 485(K, H, M, P, Q, S, T).

The productive positions in Amy 707 that fall within the previouslydescribed Productivity Scores of “2, 3 and 4” (for the full SELlibraries) and the substitutions within those positions that arecombinable are listed, below, in LIST C. Position numbering is based onthe mature Amy707 protein listed in SEQ ID NO: 3.

List C

1(H, A, C, E, F, I, K, L, M, N, Q, R, T, W); 2(H, A, C, D, E, F, G, I,K, L, M, N, P, Q, S, W); 3(N, A, C, D, E, F, K, L, M, Q, S, T, V); 4(G,D, E, F, H, I, K, L, M, P, S, T, W); 5(T, A, C, D, G, H, I, M, N, Q, S,V, W); 6(N, A, E, G, Q, S, T); 7(G, A, D, H, I, L, M, P, Q, R, S, T, V,Y); 16(Y, A, D, E, H, N, T, W); 17(L, A, D, G, S, T, V); 20(D, A, C, E,G, H, I, N, S, Y); 22(N, E, G, I, L, M, Q, R, S, T, V, W); 23(H, F, M,Q, T); 25(N, A, C, G, K, M, S, T, V, Y); 26(R, K, Q, T); 27(L, A, I, V);28(N, A, C, D, E, G, H, K, Q, R, W, Y); 29(S, A, C, D, E, F, H, K, M, N,R, T, V, W, Y); 30(D, E, M, N, Q, R); 32(S, C, D, E, G, I, L, M, N, Q,R, W, Y); 33(N, C, D, H, I, K, M, Q, R, T, V, W, Y); 35(K, A, C, E, F,G, H, I, L, M, N, Q); 36(S, D, G, K, Q, T); 41(A, C, D, I, K, M, Q, S);47(A, G, M, P, S); 50(G, C, S); 52(S, K, L, M, R, T); 54(N, A, C, D, E,F, G, M, Q, S, V, W); 56(V, E, N, S); 63(L, M, N, Q); 68(E, A, D, Q);70(N, C, D, E, F, G, H, I, K, L, M, R, S, V); 73(G, D, E, K, M, Q, R, S,T, W, Y); 74(T, G, S); 77(T, A, I, N, S, V); 81(T, A, C, D, F, I, K, N,P, S); 82(R, A, C, F, I, K, M, Q, S, V, Y); 83(S, K, M, N, Q, R, T);84(Q, C, D, E, F, K, L, M, N); 86(Q, E, H, I, K, R, T, V, W, Y); 87(A,D, K, M, T); 89(V, A, C, I); 90(T, G, M, Q, R, S); 91(S, A, E, H, K, M,N, Q, R, T, V); 94(N, A, C, D, F, G, H, K, L, M, Q, R); 95(N, A, C, D,F, G, H, I, Q, R, S, T, Y); 96(G, D, E, N); 98(Q, A, C, D, E, G, H, K,R); 99(V, A, C, I); 100(Y, C, F, I); 103(V, A, C, F, I, L, T); 110(G, A,P, S); 113(A, C, E, F, G, H, I, K, M, R, V, Y); 116(M, A, C, D, E, F, G,I, L, N, P, Q, R, T, V, W); 117(V, E, L, P, R, S, T); 118(R, D, E, G, L,Q, T, V, W); 123(N, A, C, L); 125(N, C, F, G, H, I, L, M, R, S, T, W,Y); 128(N, C, E, L, Y); 133(G, A, D, H, P, Q, S, T); 134(E, D, P, S, T,V); 135(Y, C, F, L, M, Q); 136(T, C, D, F, G, K, L, M, N, P, Q, R, Y);138(E, D, L, M, N); 139(A, C, G); 142(R, C, E, F, G, H, K, S, T, Y);144(D, E, I, K, M, S, Y); 146(P, A, C, D, E, F, G, H, M, R, S, W, Y);147(G, A, D, I, L); 149(G, A, C, D, E, F, H, K, L, P, R, V, W); 150(N,H, L, M, P, R, S); 151(T, D, E, G, H, I, L, M, Q, V); 154(S, L, R, Y);156(K, A, D, S); 158(R, A, C, K, L, N, Q); 160(Y, A, C, D, E, F, G, H,I, K, L, M, N, P, Q, R, S); 169(Q, A, C, D, E, F, G, H, I, K, M, N, S,V, W, Y); 170(S, A, C, E, K); 171(R, M, S, T); 172(R, A, C, E, G, H, M,Q, S, Y); 173(L, A, C, D, F, H, K, N, W, Y); 174(N, D, G, H, I, L, P, S,T, V); 175(N, A, D, E, L, M, R, S); 179(K, C, L, M, Q); 181(R, A, C, E,F, I, L, M, N, Q, S, T, V, Y); 183(H, A, C, D, E, F, L, M, N, P, Q, V,W, Y); 184(G, A, C, D, E, L, N); 186(A, D, E, G, N); 195(N, F, L, W, Y);206(I, C, H, M, N, S, T); 210(H, A, C, D, E, M, N, Q, R, S); 211(P, A,C, D, F, K, L, M, N, Q, S, V); 215(N, D, F, K, L, M, Q); 216(E, A, C, G,H, L, N, Q, S, T, Y); 218(R, A, C, E, F, H, K, M); 219(N, A, C, D, M, R,T); 222(V, A, D, E, F, G, H, I, L, M, N, R, S, T, Y); 225(T, A, K, R);226(N, A, D, E, K, Y); 227(T, A, E, K); 229(G, C, F, V); 231(D, E, G,T); 233(F, A, C, H, L, M, W, Y); 235(I, L, M, V); 244(S, D, E, H, N, Q);247(R, A, E, L, T); 249(W, F, L, M); 250(I, L, M, V); 251(N, A, C, G, H,K, L, P, Q, R, S, T, V, W, Y); 252(H, D, E, K, N); 257(T, A, M, S);258(G, D, I, K, M, N, P, R, S, V); 259(K, A, C, D, E, G, H, P, Q, R, T);260(N, D, K, P, R); 261(M, A, C, E, G, I, Q, T); 262(F, A, C, D, E, G,H, K, L, M, Q, R, S, Y); 273(G, C, D, E, H, K, L, M, P, Q, S, T, V, Y);280(Q, D, H, K, M, N, V); 285(N, E, K, M, S, T); 286(H, A, C, E, F, L,M, N, T, V); 287(S, A, D, N, Y); 288(V, C, I, L, T); 298(Y, F, R, W);299(N, G, H, M, R, S, T, Y); 302(K, C, E, M, Q, R, S, T); 303(S, A, C,D, E, G, L, M, Q, R); 304(G, C, K, R, S, V); 306(N, A, D, G); 311(N, D,E, F, G, H, K, L, M, Q, R, T, Y); 312(I, L, M, V); 314(N, A, D, E, G, H,I, K, L, M, Q, S, T, V, Y); 318(V, C, I, L, M, S, T); 319(Q, A, D, E, G,H, N, R, Y); 320(R, A, D, E, H, K, M, N, Q, S, T, Y); 323(S, A, C, D, E,F, G, H, I, L, M, P, R, T, V, Y); 324(H, A, C, K, M, Y); 326(V, A, C, H,M, N, T); 337(E, A, C, D, N, Q, S, T, Y); 339(A, G, S, T); 341(E, A, D,F, G, H, K, Y); 345(E, A, G, H, K, L, M, N, Q, S, T, Y); 346(E, A, C, D,G, H, K, M, N, Q, R, S, T, V, Y); 351(L, A, C, M, Q); 355(L, I, K, M,V); 356(T, C, I, L, Q, V); 357(L, A, H, M); 358(T, A, C, G, I,; 359(R,I, V, W); 360(E, A, C, F, H, K, L, N, P, Q, R, T, V, Y); 361(Q, A, C, D,E, G, H, S, T, V, W); 363(Y, A, D, E, I, K, M, N, Q, V, W); 364(P, A, C,G); 365(S, A, G, N, V); 366(V, C, I, L); 368(Y, G, L, M, Q); 372(Y, H,I, K, M, Q, R, T, V); 374(I, C, N, Q, S); 375(P, A, D, E, G, H, I, K, M,Q, R, T, V, Y); 377(H, A, G, K, M, T); 378(G, E, H, L, M, N); 379(V, A,I, L, M, N, Q, R, S, Y); 380(P, D, E, G, H, K, Q, S); 381(A, G, N, Q, R,S, T); 387(D, E, G, N); 388(P, A, C, D, F, G, H, K, L, N, Q, R, S, T, V,Y); 389(I, E, F, G, L, M, Q, S, V); 391(E, A, C, G, H, I, K, L, N, R, S,W); 392(A, C, G, S); 394(Q, C, D, E, G, H, L, R, V, Y); 395(K, A, D, E,G, M, Q, R, S, T, V); 396(Y, K, M, N); 400(K, A, F, G, H, I, L, M, Q, T,V, W); 402(N, C, I, K, L, S, V, Y); 405(L, A, C, M, N, T, V); 406(D, A,C, L, N, Q); 408(H, E, G, K, M, N, P, Q, R, S, T); 413(W, F, H, I, L,Y); 414(T, A, C, S, V); 415(R, C, W, Y); 416(E, A, D, F, G, H, K, L, N,Q, R, T, V, W, Y); 418(N, A, D, I, K, L, M, Q, S, T, V); 419(T, D, E, H,K, L, M, N, P, Q, R, S, W, Y); 420(A, D, F, G, H, I, L, M, Q, R, S, T,V, W); 421(H, A, C, D, E, I, K, L, M, N, R, V, W, Y); 422(P, A, C, E, F,G, L, M, T, V, Y); 423(N, C, D, E, F, H, I, L, R, S, T); 424(S, A, C, D,E, G, I, N, Q, T, V, W); 426(L, A, N, S); 430(M, G, I, L, V); 433(G, A,C, D, E, K, M, N, R); 434(A, C, D, E, F, H, I, K, M, N, P, Q, R, S, T,V); 435(G, A, C, E, K, M, N, P, Q, R, T); 436(G, A, C, D, Q, S); 437(S,A, C, D, K, N, T); 438(K, C, E, H, S); 439(W, H, L, M, Q); 441(F, H, N,Y); 444(R, A, K, Q); 445(N, A, C, E, G, K, Q, R, T); 446(K, A, C, F, H,M, Q, S, T, Y); 450(V, A, E, I, L, Q, R, S, T); 452(S, A, C, E, F, H, K,N, Q, T, W, Y); 454(I, A, C, F, L, M, S, V); 457(N, G, H, Q, R, T);458(R, C, D, E, H, K, L, M, N, S, T, V, Y); 459(T, A, C, D, E, G, H, L,N, P, S); 460(G, E, H, K, N, Q, S); 461(T, A, C, D, E, F, G, K, L, N, P,Q, R, V, Y); 463(T, E, K, L, P, Q, R); 465(N, D, G, Q); 466(A, D, E, G,K, N, P, Q, R, S); 471(N, C, D, E, H, Q, R, Y); 476(G, D, E, H, N, R);477(G, A, D, K, N, P, Q, R, T); 481(I, L, T, V); 483(V, C, G, H, M, R,S, T); 484(N, A, E, G, H, Q, R, S); and 485(K, H, M, P, Q, S, T).

The productive positions in Amy 707 that fall within the previouslydescribed Productivity Scores of “1, 2, 3 and 4” (for the full SELlibraries) and the substitutions within those positions that arecombinable are listed, below, in LIST D. Position numbering is based onthe mature Amy707 protein listed in SEQ ID NO: 3.

List D

1(H, A, C, E, F, I, K, L, M, N, Q, R, T, W); 2(H, A, C, D, E, F, G, I,K, L, M, N, P, Q, S, W); 3(N, A, C, D, E, F, K, L, M, Q, S, T, V); 4(G,D, E, F, H, I, K, L, M, P, S, T, W); 5(T, A, C, D, G, H, I, M, N, Q, S,V, W); 6(N, A, E, G, Q, S, T); 7(G, A, D, H, I, L, M, P, Q, R, S, T, V,Y); 10(M, I, L); 12(Y, A); 16(Y, A, D, E, H, N, T, W); 17(L, A, D, G, S,T, V); 18(P, A, E); 19(N, D, L); 20(D, A, C, E, G, H, I, N, S, Y); 22(N,E, G, I, L, M, Q, R, S, T, V, W); 23(H, F, M, Q, T); 25(N, A, C, G, K,M, S, T, V, Y); 26(R, K, Q, T); 27(L, A, I, V); 28(N, A, C, D, E, G, H,K, Q, R, W, Y); 29(S, A, C, D, E, F, H, K, M, N, R, T, V, W, Y); 30(D,E, M, N, Q, R); 31(A, S); 32(S, C, D, E, G, I, L, M, N, Q, R, W, Y);33(N, C, D, H, I, K, M, Q, R, T, V, W, Y); 34(L, F, M); 35(K, A, C, E,F, G, H, I, L, M, N, Q); 36(S, D, G, K, Q, T); 40(T, K, N); 41(A, C, D,I, K, M, Q, S); 47(A, G, M, P, S); 50(G, C, S); 52(S, K, L, M, R, T);53(Q, A); 54(N, A, C, D, E, F, G, M, Q, S, V, W); 56(V, E, N, S); 61(Y,F); 63(L, M, N, Q); 64(Y, H); 66(L, M, V); 68(E, A, D, Q); 70(N, C, D,E, F, G, H, I, K, L, M, R, S, V); 72(K, R); 73(G, D, E, K, M, Q, R, S,T, W, Y); 74(T, G, S); 75(V, I, M); 77(T, A, I, N, S, V); 81(T, A, C, D,F, I, K, N, P, S); 82(R, A, C, F, I, K, M, Q, S, V, Y); 83(S, K, M, N,Q, R, T); 84(Q, C, D, E, F, K, L, M, N); 86(Q, E, H, I, K, R, T, V, W,Y); 87(A, D, K, M, T); 88(A, M); 89(V, A, C, I); 90(T, G, M, Q, R, S);91(S, A, E, H, K, M, N, Q, R, T, V); 93(K, H, R); 94(N, A, C, D, F, G,H, K, L, M, Q, R); 95(N, A, C, D, F, G, H, I, Q, R, S, T, Y); 96(G, D,E, N); 97(I, V); 98(Q, A, C, D, E, G, H, K, R); 99(V, A, C, I); 100(Y,C, F, I); 101(G, A); 103(V, A, C, F, I, L, T); 110(G, A, P, S); 111(A,S); 112(D, C, E); 113(A, C, E, F, G, H, I, K, M, R, V, Y); 115(E, Q);116(M, A, C, D, E, F, G, I, L, N, P, Q, R, T, V, W); 117(V, E, L, P, R,S, T); 118(R, D, E, G, L, Q, T, V, W); 119(A, C, S); 122(V, C); 123(N,A, C, L); 124(P, N, T); 125(N, C, F, G, H, I, L, M, R, S, T, W, Y);126(N, D); 128(N, C, E, L, Y); 129(Q, V); 132(T, S); 133(G, A, D, H, P,Q, S, T); 134(E, D, P, S, T, V); 135(Y, C, F, L, M, Q); 136(T, C, D, F,G, K, L, M, N, P, Q, R, Y); 138(E, D, L, M, N); 139(A, C, G); 140(W, F,Y); 142(R, C, E, F, G, H, K, S, T, Y); 144(D, E, I, K, M, S, Y); 145(F,M, Y); 146(P, A, C, D, E, F, G, H, M, R, S, W, Y); 147(G, A, D, I, L);149(G, A, C, D, E, F, H, K, L, P, R, V, W); 150(N, H, L, M, P, R, S);151(T, D, E, G, H, I, L, M, Q, V); 153(S, N); 154(S, L, R, Y); 155(F,W); 156(K, A, D, S); 158(R, A, C, K, L, N, Q); 160(Y, A, C, D, E, F, G,H, I, K, L, M, N, P, Q, R, S); 162(F, M); 165(V, C, T); 167(W, F, M);168(D, C); 169(Q, A, C, D, E, F, G, H, I, K, M, N, S, V, W, Y); 170(S,A, C, E, K); 171(R, M, S, T); 172(R, A, C, E, G, H, M, Q, S, Y); 173(L,A, C, D, F, H, K, N, W, Y); 174(N, D, G, H, I, L, P, S, T, V); 175(N, A,D, E, L, M, R, S); 176(R, K, T); 178(Y, W); 179(K, C, L, M, Q); 181(R,A, C, E, F, I, L, M, N, Q, S, T, V, Y); 182(G, C, D); 183(H, A, C, D, E,F, L, M, N, P, Q, V, W, Y); 184(G, A, C, D, E, L, N); 186(A, D, E, G,N); 195(N, F, L, W, Y); 196(G, C); 203(Y, N); 206(I, C, H, M, N, S, T);210(H, A, C, D, E, M, N, Q, R, S); 211(P, A, C, D, F, K, L, M, N, Q, S,V); 215(N, D, F, K, L, M, Q); 216(E, A, C, G, H, L, N, Q, S, T, Y);217(L, M); 218(R, A, C, E, F, H, K, M); 219(N, A, C, D, M, R, T); 221(G,I, V); 222(V, A, D, E, F, G, H, I, L, M, N, R, S, T, Y); 225(T, A, K,R); 226(N, A, D, E, K, Y); 227(T, A, E, K); 228(L, I, M); 229(G, C, F,V); 230(L, M); 231(D, E, G, T); 233(F, A, C, H, L, M, W, Y); 235(I, L,M, V); 238(V, I); 243(Y, F); 244(S, D, E, H, N, Q); 245(F, E, M); 247(R,A, E, L, T); 249(W, F, L, M); 250(I, L, M, V); 251(N, A, C, G, H, K, L,P, Q, R, S, T, V, W, Y); 252(H, D, E, K, N); 253(V, M); 257(T, A, M, S);258(G, D, I, K, M, N, P, R, S, V); 259(K, A, C, D, E, G, H, P, Q, R, T);260(N, D, K, P, R); 261(M, A, C, E, G, I, Q, T); 262(F, A, C, D, E, G,H, K, L, M, Q, R, S, Y); 263(A, S); 265(A, G, S); 273(G, C, D, E, H, K,L, M, P, Q, S, T, V, Y); 276(E, C, T); 280(Q, D, H, K, M, N, V); 283(N,D, G); 285(N, E, K, M, S, T); 286(H, A, C, E, F, L, M, N, T, V); 287(S,A, D, N, Y); 288(V, C, I, L, T); 292(P, L, M); 296(N, Q); 297(L, M);298(Y, F, R, W); 299(N, G, H, M, R, S, T, Y); 301(S, A, G); 302(K, C, E,M, Q, R, S, T); 303(S, A, C, D, E, G, L, M, Q, R); 304(G, C, K, R, S,V); 306(N, A, D, G); 307(Y, A, F); 310(R, Q, S); 311(N, D, E, F, G, H,K, L, M, Q, R, T, Y); 312(I, L, M, V); 313(F, M, Y); 314(N, A, D, E, G,H, I, K, L, M, Q, S, T, V, Y); 317(V, L); 318(V, C, I, L, M, S, T);319(Q, A, D, E, G, H, N, R, Y); 320(R, A, D, E, H, K, M, N, Q, S, T, Y);321(H, W, Y); 322(P, D); 323(S, A, C, D, E, F, G, H, I, L, M, P, R, T,V, Y); 324(H, A, C, K, M, Y); 326(V, A, C, H, M, N, T); 327(T, L);328(F, V); 329(V, I); 334(S, T); 337(E, A, C, D, N, Q, S, T, Y); 339(A,G, S, T); 341(E, A, D, F, G, H, K, Y); 343(F, T, Y); 344(V, C, I);345(E, A, G, H, K, L, M, N, Q, S, T, Y); 346(E, A, C, D, G, H, K, M, N,Q, R, S, T, V, Y); 347(W, A, D); 350(P, E); 351(L, A, C, M, Q); 352(A,S); 354(A, S); 355(L, I, K, M, V); 356(T, C, I, L, Q, V); 357(L, A, H,M); 358(T, A, C, G, I,; 359(R, I, V, W); 360(E, A, C, F, H, K, L, N, P,Q, R, T, V, Y); 361(Q, A, C, D, E, G, H, S, T, V, W); 363(Y, A, D, E, I,K, M, N, Q, V, W); 364(P, A, C, G); 365(S, A, G, N, V); 366(V, C, I, L);367(F, Y); 368(Y, G, L, M, Q); 369(G, A, S); 372(Y, H, I, K, M, Q, R, T,V); 374(I, C, N, Q, S); 375(P, A, D, E, G, H, I, K, M, Q, R, T, V, Y);377(H, A, G, K, M, T); 378(G, E, H, L, M, N); 379(V, A, I, L, M, N, Q,R, S, Y); 380(P, D, E, G, H, K, Q, S); 381(A, G, N, Q, R, S, T); 382(M,K); 386(I, L, V); 387(D, E, G, N); 388(P, A, C, D, F, G, H, K, L, N, Q,R, S, T, V, Y); 389(I, E, F, G, L, M, Q, S, V); 390(L, M, V); 391(E, A,C, G, H, I, K, L, N, R, S, W); 392(A, C, G, S); 394(Q, C, D, E, G, H, L,R, V, Y); 395(K, A, D, E, G, M, Q, R, S, T, V); 396(Y, K, M, N); 397(A,G); 400(K, A, F, G, H, I, L, M, Q, T, V, W); 401(Q, H, M); 402(N, C, I,K, L, S, V, Y); 403(D, E, T); 405(L, A, C, M, N, T, V); 406(D, A, C, L,N, Q); 408(H, E, G, K, M, N, P, Q, R, S, T); 410(I, N); 411(I, V);412(G, A, S); 413(W, F, H, I, L, Y); 414(T, A, C, S, V); 415(R, C, W,Y); 416(E, A, D, F, G, H, K, L, N, Q, R, T, V, W, Y); 417(G, A); 418(N,A, D, I, K, L, M, Q, S, T, V); 419(T, D, E, H, K, L, M, N, P, Q, R, S,W, Y); 420(A, D, F, G, H, I, L, M, Q, R, S, T, V, W); 421(H, A, C, D, E,I, K, L, M, N, R, V, W, Y); 422(P, A, C, E, F, G, L, M, T, V, Y); 423(N,C, D, E, F, H, I, L, R, S, T); 424(S, A, C, D, E, G, I, N, Q, T, V, W);425(G, A); 426(L, A, N, S); 427(A, C, T); 428(T, N, S); 429(I, M);430(M, G, I, L, V); 431(S, A, C); 433(G, A, C, D, E, K, M, N, R); 434(A,C, D, E, F, H, I, K, M, N, P, Q, R, S, T, V); 435(G, A, C, E, K, M, N,P, Q, R, T); 436(G, A, C, D, Q, S); 437(S, A, C, D, K, N, T); 438(K, C,E, H, S); 439(W, H, L, M, Q); 441(F, H, N, Y); 442(V, A, C); 444(R, A,K, Q); 445(N, A, C, E, G, K, Q, R, T); 446(K, A, C, F, H, M, Q, S, T,Y); 448(G, F, N); 450(V, A, E, I, L, Q, R, S, T); 451(W, F); 452(S, A,C, E, F, H, K, N, Q, T, W, Y); 454(I, A, C, F, L, M, S, V); 457(N, G, H,Q, R, T); 458(R, C, D, E, H, K, L, M, N, S, T, V, Y); 459(T, A, C, D, E,G, H, L, N, P, S); 460(G, E, H, K, N, Q, S); 461(T, A, C, D, E, F, G, K,L, N, P, Q, R, V, Y); 463(T, E, K, L, P, Q, R); 465(N, D, G, Q); 466(A,D, E, G, K, N, P, Q, R, S); 467(D, E); 469(W, Y); 471(N, C, D, E, H, Q,R, Y); 473(S, P); 474(V, S); 475(N, D, E); 476(G, D, E, H, N, R); 477(G,A, D, K, N, P, Q, R, T); 478(S, A, G); 479(V, T); 481(I, L, T, V);482(W, Y); 483(V, C, G, H, M, R, S, T); 484(N, A, E, G, H, Q, R, S); and485(K, H, M, P, Q, S, T).

Although the foregoing mutations were identified using SEL librariesbased on Amy707 (SEQ ID NO: 3), it is known that many bacterial (andother) α-amylases share the same fold, and often share significant aminoacid sequence identity, and often benefit from the same mutations. Inthe present case, corresponding amino acid positions in other α-amylasescan readily be identified by amino acid sequence alignment with Amy707(SEQ ID NO: 7) using Clustal W with default parameters. α-amylases inwhich the foregoing mutations are likely to produce a performancebenefit include those having a similar fold and/or having 60% or greateramino acid sequence identity to any of the well-known Bacillus amylases(e.g., from B. lichenifomis, B. stearothermophilus, and B.amyloliquifaciens), Carbohydrate-Active Enzymes database (CAZy) Family13 amylases, or any amylase that has heretofore been referred to by thedescriptive term, “Termamyl-like.” The reader will appreciate that wherean α-amylase naturally has a mutation listed above (i.e., where thewild-type α-amylase already comprised a residue identified as amutation), then that particular mutation does not apply to thatα-amylase. However, other described mutations may work in combinationwith the naturally occuring residue at that position. Because of theirclose sequence identity (over 95%; FIG. 1), and the fact that both areG6 amylases, mutations (including substitutions, insertions, anddeletions, that produce a beneficial effect in Amy707 are likely toproduce a similar effect in AA560 amylase, and vice versa.

In some embodiments, the present α-amylase variants have at least onecombinable mutation at a productive position corresponding to thecombinable mutations at productive positions described, above, in ListsA, B, C, and/or D, and/or a combinable mutation as described in Table Cor D (which use SEQ ID NO: 3 for numbering) and a defined degree ofamino acid sequence homology/identity to SEQ ID NO: 3 or SEQ ID NO: 4,for example, at least 60%, at least 65%, at least 70%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or even at least 99% amino acidsequence homology/identity. In some embodiments, the suitability scoreof the at least one mutation is +++, ++++, or +++++. In someembodiments, the suitability score of the at least one mutation is ++++,or +++++. In some embodiments, the suitability score of the at least onemutation is +++++. In some embodiments, the variants have a plurality(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, or more) combinable mutations.

In some embodiments, the present α-amylase variants have at least onecombinable mutation at a productive position corresponding to thecombinable mutations at productive positions described, above, in ListsA, B, C, and/or D, and/or a combinable mutation as described in Table Cor D (which use SEQ ID NO: 3 for numbering) and are derived from aparental amylase having a defined degree of amino acid sequencehomology/identity to SEQ ID NO: 3 or SEQ ID NO: 4, for example, at least60%, at least 65%, at least 70%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98% or even at least 99% amino acid sequencehomology/identity. In some embodiments, the suitability score of the atleast one mutation is +++, ++++, or +++++. In some embodiments, thesuitability score of the at least one mutation is ++++, or +++++. Insome embodiments, the suitability score of the at least one mutation is+++++. In some embodiments, the variants have, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more combinablemutations.

2.2 Additional Mutations

In some embodiments, in addition to one or more of the mutationsdescribed above (e.g., in Section 2.1), the present amylases furtherinclude one or more mutations that provide a further performance orstability benefit. Exemplary performance benfits include but are notlimited to increased hydrolysis of a starch substrate, increased grain,cereal or other starch substrate liquifaction performance, increasedcleaning performance, increased thermal stability, increased storagestability, increased solubility, an altered pH profile, decreasedcalcium dependence, increased specific activity, modified substratespecificity, modified substrate binding, modified pH-dependent activity,modified pH-dependent stability, increased oxidative stability, andincreased expression. In some cases, the performance benefit is realizedat a relatively low temperature. In some cases, the performance benefitis realized at relatively high temperature.

In some embodiments, the present α-amylase variants additionally have atleast one mutation in the calcium binding loop based on the work ofSuzuki et al. (1989) J. Biol. Chem. 264:18933-938. Exemplary mutationsinclude a deletion or substitution at one or more residues correspondingto Arg-181, Gly-182, His-183, or Gly-184 in SEQ ID NO: 3. In particularembodiments, the mutation corresponds to the deletion of Arg-181 andGly-182 or His-183 and Gly-184 (using SEQ ID NO: 3 numbering).Homologous residues in other amylases can be determined by structuralalignment, or by primary structure alignment.

In some embodiments, the present α-amylase variants additionally have atleast one mutation known to produce a performance, stability, orsolubility benefit in other microbial α-amylases, including but notlimited to those having a similar fold and/or having 60% or greateramino acid sequence identity to Amy707 (SEQ ID NO: 3) or AA560 (SEQ IDNO: 4), any of the well-known Bacillus amylases (e.g., from B.lichenifomis, B. stearothermophilus, and B. amyloliquifaciens),Carbohydrate-Active Enzymes database (CAZy) Family 13 amylases, or anyamylase that has heretofore been referred to by the descriptive term,“Termamyl-like.” Amino acid sequence identity can be determined usingClustal W with default parameters.

Furthermore, the present amylases may include any number of conservativeamino acid substitutions. Exemplary conservative amino acidsubstitutions are listed in the Table E.

TABLE E Conservative amino acid substitutions For Amino Acid CodeReplace with any of Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-CysArginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met,D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-GlnAspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine CD-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn,Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn,Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, b-Ala, Acp Isoleucine ID-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val,Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg,Met, D- Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile,D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa,His, D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or5-phenylproline Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid,D-or L-1- oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met(O), D- Met(O), L-Cys, D-Cys Threonine T D-Thr,Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val TyrosineY D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,D-Ile, Met, D-Met

The reader will appreciate that some of the above mentioned conservativemutations can be produced by genetic manpulation, while others areproduced by introducing synthetic amino acids into a polypeptide bygenetic or other means.

The present amylase may be “precursor,” “immature,” or “full-length,” inwhich case they include a signal sequence, or “mature,” in which casethey lack a signal sequence. Mature forms of the polypeptides aregenerally the most useful. Unless otherwise noted, the amino acidresidue numbering used herein refers to the mature forms of therespective amylase polypeptides. The present amylase polypeptides mayalso be truncated to remove the N or C-termini, so long as the resultingpolypeptides retain amylase activity.

The present amylase may be a “chimeric” or “hybrid” polypeptide, in thatit includes at least a portion of a first amylase polypeptide, and atleast a portion of a second amylase polypeptide (such chimeric amylaseshave recently been “rediscovered” as domain-swap amylases). The presentamylases may further include heterologous signal sequence, an epitope toallow tracking or purification, or the like. Exemplary heterologoussignal sequences are from B. licheniformis amylase (LAT), B. subtilis(AmyE or AprE), and Streptomyces CelA.

2.3. Nucleotides Encoding Variant Amylase Polypeptides

In another aspect, nucleic acids encoding a variant amylase polypeptideare provided. The nucleic acid may encode a particular amylasepolypeptide, or an amylase having a specified degree of amino acidsequence identity to the particular amylase.

In one example, the nucleic acid encodes an amylase having at least 60%,at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or even at least 99% homology/identity to SEQ ID NO: 3 or SEQID NO: 4 (excluding the portion of the nucleic acid that encodes thesignal sequence). It will be appreciated that due to the degeneracy ofthe genetic code, a plurality of nucleic acids may encode the samepolypeptide.

In another example, the nucleic acid hybridizes under stringent or verystringent conditions to a nucleic acid encoding (or complementary to anucleic acid encoding) an amylase having at least 60%, at least 65%, atleast 70%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% oreven at least 99% homology/identity to SEQ ID NO: 3 or SEQ ID NO: 4(excluding the portion of the nucleic acid that encodes the signalsequence). Such hybridization conditions are described herein but arealso well known in the art.

In a particular example, the nucleic acid hybridizes under stringent orvery stringent conditions to the nucleic acid of SEQ ID NO: 1 or SEQ IDNO: 5, which are shown, below.

SEQ ID NO: 1: Codon optimized nucleic acid encoding SEQ ID NO: 3.ATGAAACAACAAAAACGGCTTTACGCCCGATTGCTGACGCTGTTATTTGCGCTCATCTTCTTGCTGCCTCATTCTGCAGCTTCAGCACATCATAATGGCACAAACGGCACGATGATGCAGTATTTTGAATGGTATCTGCCGAACGATGGAAACCATTGGAACCGCCTGAATAGCGATGCGAGCAACCTGAAAAGCAAAGGCATCACAGCAGTTTGGATTCCGCCGGCATGGAAAGGAGCAAGCCAAAACGACGTCGGCTATGGAGCGTATGATCTGTATGACCTGGGCGAATTTAACCAAAAAGGCACGGTCCGCACGAAATATGGCACGCGCAGCCAACTTCAAGCAGCAGTCACGAGCCTTAAAAACAACGGCATCCAGGTCTATGGAGATGTCGTCATGAACCATAAAGGCGGAGCAGATGCGACAGAAATGGTCAGAGCGGTCGAAGTCAACCCGAACAACCGCAATCAAGAAGTCACGGGCGAATATACAATCGAAGCGTGGACGCGCTTTGATTTTCCGGGCAGAGGCAATACACATAGCAGCTTTAAATGGCGCTGGTATCATTTTGATGGCGTCGATTGGGATCAAAGCCGCAGACTGAACAACCGCATCTATAAATTTCGCGGCCATGGCAAAGCATGGGATTGGGAAGTCGATACGGAAAACGGCAACTATGACTATCTGATGTATGCGGACATCGATATGGATCATCCGGAAGTCGTCAACGAACTGAGAAATTGGGGCGTCTGGTATACAAATACGCTGGGCCTGGATGGCTTTAGAATCGACGCGGTCAAACATATCAAATATAGCTTTACGCGCGACTGGATCAATCATGTCAGAAGCGCGACGGGCAAAAATATGTTTGCGGTCGCGGAATTTTGGAAAAATGATCTGGGCGCGATCGAAAACTATCTGCAAAAAACGAACTGGAACCATAGCGTCTTTGATGTCCCGCTGCATTATAACCTGTATAACGCGAGCAAAAGCGGCGGCAATTATGATATGCGCAACATCTTTAACGGCACGGTCGTTCAAAGACATCCGAGCCATGCGGTCACGTTTGTCGATAACCATGATAGCCAACCGGAAGAAGCGCTGGAAAGCTTTGTCGAAGAATGGTTTAAACCGCTGGCGTATGCACTGACACTGACGAGAGAACAAGGATATCCGAGCGTCTTTTATGGCGACTATTATGGCATCCCGACACATGGAGTTCCGGCGATGAGAAGCAAAATCGACCCGATCCTGGAAGCGAGACAGAAATATGCGTATGGCAAACAGAACGACTATCTGGACCATCATAACATCATCGGCTGGACGAGAGAAGGAAATACGGCGCATCCGAATTCAGGACTGGCGACGATTATGTCAGATGGAGCGGGCGGAAGCAAATGGATGTTTGTCGGCAGAAACAAAGCAGGACAAGTCTGGAGCGATATCACGGGCAATAGAACGGGAACGGTCACGATCAATGCAGATGGCTGGGGCAACTTTAGCGTTAATGGCGGAAGCGTCAGCATCTGGGTCAACAAA SEQ ID NO: 5: GenebankAccession No. M18862 GGATCCCGTCTACGGAGAAGCGAGTATTGAATTTTTTGCTGTAACAGAAAGCGAGCGTGGGAAAGGATTTGGCTTTCAATTACTAACGGTTGCTTTAAATTGGCTATTTACGATTGATACGATTCATTCAATTACACTCTGTGTCGATTCTAGTAATGAACATGCGATTCATTTATATAAAAAAGTTGGATTCAGGCATGTTCATGATTTGAGTTATTTTACTAAAGAAGTATCTCATTAAAAACATGATTGAGGAAAGACGGTTTTCGACTAATTGTGGTCAAAGTAGAAAATTGAATGAATATTACGAAGCATGAGGCTAAGACATAACTAAAGTGTCTAAATGAAAAACCGAACGAAAAATGAACGAAGCGAAGTGTATTTCAAGAAAGGTTACCGTTCGCTATTTATCACCGTTCGGTTATTTTTTAGATAAGCCACTTTTGTCGCGGCCTCTTTTTGGTGCCGATAAATGAGAATAAAGAATAAAAAGTCAATATTGCTTAGCTAAATGAATGTCAAGGTGGTTATATTATCCTATTTATTTTCAGAAAATAAAAAAACGTTTGCGCAATTGTTTTATAGCATAATAATATAACCTTGCCAATTGATATTTAAGTCGAGTGAAATCAATTGCGCAAATTAATGAGTGTGTTCAAGGAGAGTGATGAATGTAGCAGTTTAGTCATGTACTTGTTTTTGGAAAGCGCTTACAATTAGGAGGGTGGATGAAAATGAGAACAGGAAAAAAGGGTTTTTTAAGTATTTTATTAGCGTTCTTATTGGTGATTACTTCAATACCGTTTACTTTAGTAGATGTAGAAGCACATCATAACGGTACGAACGGGACAATGATGCAATACTTTGAATGGTATCTACCTAATGACGGAAATCATTGGAATCGATTAAACTCTGATGCGAGTAACCTTAAAAGCAAAGGGATTACAGCGGTGTGGATTCCTCCAGCATGGAAGGGCGCTTCTCAAAATGACGTAGGATACGGAGCCTATGACCTGTATGATCTGGGAGAATTTAATCAAAAAGGTACCGTCCGTACAAAATATGGAACACGTAGTCAGTTACAAGCTGCGGTAACCTCCTTAAAAAATAATGGAATTCAAGTATATGGTGACGTTGTTATGAATCACAAAGGTGGCGCAGACGCTACTGAAATGGTAAGGGCCGTTGAAGTGAATCCCAATAACCGTAACCAAGAAGTGACTGGTGAATATACCATTGAAGCTTGGACTAGATTTGATTTTCCAGGGCGAGGAAATACTCATTCTAGCTTTAAATGGAGATGGTATCATTTTGATGGTGTGGATTGGGATCAGTCACGTAGACTGAACAATCGCATCTATAAATTTAGAGGTCATGGCAAAGCTTGGGATTGGGAAGTTGATACGGAAAATGGTAATTATGATTATTTAATGTACGCTGATATTGATATGGATCACCCAGAAGTAGTAAATGAATTAAGAAATTGGGGTGTTTGGTACACAAACACATTAGGACTCGATGGATTTAGAATAGATGCGGTTAAACATATAAAGTATAGCTTTACGCGCGATTGGATTAATCACGTTAGAAGTGCAACAGGTAAAAATATGTTTGCGGTTGCTGAGTTTTGGAAGAATGATTTAGGTGCAATTGAAAACTATCTGCAGAAAACAAACTGGAACCATTCAGTCTTTGATGTGCCGTTACATTATAATCTTTATAATGCATCAAAAAGCGGAGGGAACTATGATATGCGAAACATATTTAATGGAACGGTTGTTCAACGACATCCAAGTCATGCTGTAACATTTGTTGATAATCATGATTCGCAGCCTGAAGAAGCATTAGAATCTTTTGTTGAAGAATGGTTTAAACCATTAGCGTATGCGCTTACATTAACGCGTGAACAAGGATACCCTTCTGTATTTTACGGAGATTATTATGGGATTCCAACACATGGAGTGCCAGCAATGAGATCAAAAATCGATCCGATTTTAGAAGCACGTCAAAAGTATGCATACGGAAAACAAAATGATTACTTAGACCATCATAATATCATTGGTTGGACGCGTGAAGGGAATACAGCACACCCCAATTCAGGTCTAGCTACCATCATGTCTGATGGAGCGGGTGGAAGTAAGTGGATGTTTGTTGGGCGTAATAAGGCTGGTCAAGTATGGAGTGATATTACAGGAAACCGTACAGGTACGGTTACAATCAATGCAGACGGTTGGGGCAATTTCTCTGTGAATGGAGGGTCAGTTTCTATTTGGGTCAACAAATAAAAGTGGAAAAGAAGAGGCCGTAGGTTAATATGGTCTTTTCTTTTCTTTTAAGGAGGTTCAATGAATTTGTCGGTTATCCAATTATTACATGCTGAGCTGTTAGATTATTCGT

Nucleic acids may encode a “full-length” (“fl” or “FL”) amylase, whichincludes a signal sequence, only the mature form of an amylase, whichlacks the signal sequence, or a truncated form of an amylase, whichlacks the N or C-terminus of the mature form.

A nucleic acid that encodes a α-amylase can be operably linked tovarious promoters and regulators in a vector suitable for expressing theα-amylase in host cells. Exemplary promoters are from B. licheniformisamylase (LAT), B. subtilis (AmyE or AprE), and Streptomyces CelA. Such anucleic acid can also be linked to other coding sequences, e.g., toencode a chimeric polypeptide.

3. Production of Variant Amylases

The present variant amylases can be produced in host cells, for example,by secretion or intracellular expression. A cultured cell material(e.g., a whole-cell broth) comprising a variant amylase can be obtainedfollowing secretion of the variant amylase into the cell medium.Optionally, the variant amylase can be isolated from the host cells, oreven isolated from the cell broth, depending on the desired purity ofthe final variant amylase. A gene encoding a variant amylase can becloned and expressed according to methods well known in the art.Suitable host cells include bacterial, fungal (including yeast andfilamentous fungi), and plant cells (including algae). Particularlyuseful host cells include Aspergillus niger, Aspergillus oryzae orTrichoderma reesei. Other host cells include bacterial cells, e.g.,Bacillus subtilis or B. licheniformis, as well as Streptomyces.

The host cell further may express a nucleic acid encoding a homologousor heterologous glucoamylase, i.e., a glucoamylase that is not the samespecies as the host cell, or one or more other enzymes. The glucoamylasemay be a variant glucoamylase, such as one of the glucoamylase variantsdisclosed in U.S. Pat. No. 8,058,033 (Danisco US Inc.), for example.Additionally, the host may express one or more accessory enzymes,proteins, peptides. These may benefit liquefaction, saccharification,fermentation, SSF, etc processes. Furthermore, the host cell may producebiochemicals in addition to enzymes used to digest the variousfeedstock(s). Such host cells may be useful for fermentation orsimultaneous saccharification and fermentation processes to reduce oreliminate the need to add enzymes.

3.1. Vectors

A DNA construct comprising a nucleic acid encoding variant amylases canbe constructed to be expressed in a host cell. Representative nucleicacids that encode variant amylases include SEQ ID NO: 4. Because of thewell-known degeneracy in the genetic code, variant polynucleotides thatencode an identical amino acid sequence can be designed and made withroutine skill. It is also well-known in the art to optimize codon usefor a particular host cell. Nucleic acids encoding variant amylases canbe incorporated into a vector. Vectors can be transferred to a host cellusing well-known transformation techniques, such as those disclosedbelow.

The vector may be any vector that can be transformed into and replicatedwithin a host cell. For example, a vector comprising a nucleic acidencoding a variant amylase can be transformed and replicated in abacterial host cell as a means of propagating and amplifying the vector.The vector also may be transformed into an expression host, so that theencoding nucleic acids can be expressed as a functional amylase. Hostcells that serve as expression hosts can include filamentous fungi, forexample. The Fungal Genetics Stock Center (FGSC) Catalogue of Strainslists suitable vectors for expression in fungal host cells. See FGSC,Catalogue of Strains, University of Missouri, at www.fgsc.net (lastmodified Jan. 17, 2007). A representative vector is pJG153, apromoterless Cre expression vector that can be replicated in a bacterialhost. See Harrison et al. (June 2011) Applied Environ. Microbiol. 77:3916-22. pJG153 can be modified with routine skill to comprise andexpress a nucleic acid encoding an amylase variant.

A nucleic acid encoding a variant amylase can be operably linked to asuitable promoter, which allows transcription in the host cell. Thepromoter may be any DNA sequence that shows transcriptional activity inthe host cell of choice and may be derived from genes encoding proteinseither homologous or heterologous to the host cell. Exemplary promotersfor directing the transcription of the DNA sequence encoding a variantamylase, especially in a bacterial host, are the promoter of the lacoperon of E. coli, the Streptomyces coelicolor agarase gene dagA or celApromoters, the promoters of the Bacillus licheniformis α-amylase gene(amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amyM), the promoters of the Bacillus amyloliquefaciensα-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylBgenes etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral α-amylase, A. niger acid stable α-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase, or A. nidulans acetamidase. When agene encoding an amylase is expressed in a bacterial species such as E.coli, a suitable promoter can be selected, for example, from abacteriophage promoter including a T7 promoter and a phage lambdapromoter. Examples of suitable promoters for the expression in a yeastspecies include but are not limited to the Gal 1 and Gal 10 promoters ofSaccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters.cbh1 is an endogenous, inducible promoter from T. reesei. See Liu et al.(2008) “Improved heterologous gene expression in Trichoderma reesei bycellobiohydrolase I gene (cbh1) promoter optimization,” Acta Biochim.Biophys. Sin (Shanghai) 40(2): 158-65.

The coding sequence can be operably linked to a signal sequence. The DNAencoding the signal sequence may be the DNA sequence naturallyassociated with the amylase gene to be expressed or from a differentGenus or species. A signal sequence and a promoter sequence comprising aDNA construct or vector can be introduced into a fungal host cell andcan be derived from the same source. For example, the signal sequence isthe cbh1 signal sequence that is operably linked to a cbh1 promoter.

An expression vector may also comprise a suitable transcriptionterminator and, in eukaryotes, polyadenylation sequences operably linkedto the DNA sequence encoding a variant amylase. Termination andpolyadenylation sequences may suitably be derived from the same sourcesas the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell. Examples of such sequences are the originsof replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1, and0.1702.

The vector may also comprise a selectable marker, e.g., a gene theproduct of which complements a defect in the isolated host cell, such asthe dal genes from B. subtilis or B. licheniformis, or a gene thatconfers antibiotic resistance such as, e.g., ampicillin, kanamycin,chloramphenicol or tetracycline resistance. Furthermore, the vector maycomprise Aspergillus selection markers such as amdS, argB, niaD andxxsC, a marker giving rise to hygromycin resistance, or the selectionmay be accomplished by co-transformation, such as known in the art. Seee.g., International PCT Application WO 91/17243.

Intracellular expression may be advantageous in some respects, e.g.,when using certain bacteria or fungi as host cells to produce largeamounts of amylase for subsequent enrichment or purification.Extracellular secretion of amylase into the culture medium can also beused to make a cultured cell material comprising the isolated amylase.

The expression vector typically includes the components of a cloningvector, such as, for example, an element that permits autonomousreplication of the vector in the selected host organism and one or morephenotypically detectable markers for selection purposes. The expressionvector normally comprises control nucleotide sequences such as apromoter, operator, ribosome binding site, translation initiation signaland optionally, a repressor gene or one or more activator genes.Additionally, the expression vector may comprise a sequence coding foran amino acid sequence capable of targeting the amylase to a host cellorganelle such as a peroxisome, or to a particular host cellcompartment. Such a targeting sequence includes but is not limited tothe sequence, SKL. For expression under the direction of controlsequences, the nucleic acid sequence of the amylase is operably linkedto the control sequences in proper manner with respect to expression.

The procedures used to ligate the DNA construct encoding an amylase, thepromoter, terminator and other elements, respectively, and to insertthem into suitable vectors containing the information necessary forreplication, are well known to persons skilled in the art (see, e.g.,Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2^(nd) ed.,Cold Spring Harbor, 1989, and 3^(rd) ed., 2001).

3.2. Transformation and Culture of Host Cells

An isolated cell, either comprising a DNA construct or an expressionvector, is advantageously used as a host cell in the recombinantproduction of an amylase. The cell may be transformed with the DNAconstruct encoding the enzyme, conveniently by integrating the DNAconstruct (in one or more copies) in the host chromosome. Thisintegration is generally considered to be an advantage, as the DNAsequence is more likely to be stably maintained in the cell. Integrationof the DNA constructs into the host chromosome may be performedaccording to conventional methods, e.g., by homologous or heterologousrecombination. Alternatively, the cell may be transformed with anexpression vector as described above in connection with the differenttypes of host cells.

Examples of suitable bacterial host organisms are Gram positivebacterial species such as Bacillaceae including Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus(formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillusmegaterium, and Bacillus thuringiensis; Streptomyces species such asStreptomyces murinus; lactic acid bacterial species includingLactococcus sp. such as Lactococcus lactis; Lactobacillus sp. includingLactobacillus reuteri; Leuconostoc sp.; Pediococcus sp.; andStreptococcus sp. Alternatively, strains of a Gram negative bacterialspecies belonging to Enterobacteriaceae including E. coli, or toPseudomonadaceae can be selected as the host organism.

A suitable yeast host organism can be selected from thebiotechnologically relevant yeasts species such as but not limited toyeast species such as Pichia sp., Hansenula sp., or Kluyveromyces,Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces,including Saccharomyces cerevisiae or a species belonging toSchizosaccharomyces such as, for example, S. pombe species. A strain ofthe methylotrophic yeast species, Pichia pastoris, can be used as thehost organism. Alternatively, the host organism can be a Hansenulaspecies. Suitable host organisms among filamentous fungi include speciesof Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillustubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively,strains of a Fusarium species, e.g., Fusarium oxysporum or of aRhizomucor species such as Rhizomucor miehei can be used as the hostorganism. Other suitable strains include Thermomyces and Mucor species.In addition, Trichoderma sp. can be used as a host. A suitable procedurefor transformation of Aspergillus host cells includes, for example, thatdescribed in EP 238023. An amylase expressed by a fungal host cell canbe glycosylated, i.e., will comprise a glycosyl moiety. Theglycosylation pattern can be the same or different as present in thewild-type amylase. The type and/or degree of glycosylation may impartchanges in enzymatic and/or biochemical properties.

It is advantageous to delete genes from expression hosts, where the genedeficiency can be cured by the transformed expression vector. Knownmethods may be used to obtain a fungal host cell having one or moreinactivated genes. Gene inactivation may be accomplished by complete orpartial deletion, by insertional inactivation or by any other means thatrenders a gene nonfunctional for its intended purpose, such that thegene is prevented from expression of a functional protein. Any gene froma Trichoderma sp. or other filamentous fungal host that has been clonedcan be deleted, for example, cbh1, cbh2, egl1, and egl2 genes. Genedeletion may be accomplished by inserting a form of the desired gene tobe inactivated into a plasmid by methods known in the art.

Introduction of a DNA construct or vector into a host cell includestechniques such as transformation; electroporation; nuclearmicroinjection; transduction; transfection, e.g., lipofection mediatedand DEAE-Dextrin mediated transfection; incubation with calciumphosphate DNA precipitate; high velocity bombardment with DNA-coatedmicroprojectiles; and protoplast fusion. General transformationtechniques are known in the art. See, e.g., Sambrook et al. (2001),supra. The expression of heterologous protein in Trichoderma isdescribed, for example, in U.S. Pat. No. 6,022,725. Reference is alsomade to Cao et al. (2000) Science 9:991-1001 for transformation ofAspergillus strains. Genetically stable transformants can be constructedwith vector systems whereby the nucleic acid encoding an amylase isstably integrated into a host cell chromosome. Transformants are thenselected and purified by known techniques.

The preparation of Trichoderma sp. for transformation, for example, mayinvolve the preparation of protoplasts from fungal mycelia. See Campbellet al. (1989) Curr. Genet. 16: 53-56. The mycelia can be obtained fromgerminated vegetative spores. The mycelia are treated with an enzymethat digests the cell wall, resulting in protoplasts. The protoplastsare protected by the presence of an osmotic stabilizer in the suspendingmedium. These stabilizers include sorbitol, mannitol, potassiumchloride, magnesium sulfate, and the like. Usually the concentration ofthese stabilizers varies between 0.8 M and 1.2 M, e.g., a 1.2 M solutionof sorbitol can be used in the suspension medium.

Uptake of DNA into the host Trichoderma sp. strain depends upon thecalcium ion concentration. Generally, between about 10-50 mM CaCl₂ isused in an uptake solution. Additional suitable compounds include abuffering system, such as TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) or10 mM MOPS, pH 6.0 and polyethylene glycol. The polyethylene glycol isbelieved to fuse the cell membranes, thus permitting the contents of themedium to be delivered into the cytoplasm of the Trichoderma sp. strain.This fusion frequently leaves multiple copies of the plasmid DNAintegrated into the host chromosome.

Usually transformation of Trichoderma sp. uses protoplasts or cells thathave been subjected to a permeability treatment, typically at a densityof 10⁵ to 10⁷/mL, particularly 2×10⁶/mL. A volume of 100 μL of theseprotoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitoland 50 mM CaCl₂) may be mixed with the desired DNA. Generally, a highconcentration of PEG is added to the uptake solution. From 0.1 to 1volume of 25% PEG 4000 can be added to the protoplast suspension;however, it is useful to add about 0.25 volumes to the protoplastsuspension. Additives, such as dimethyl sulfoxide, heparin, spermidine,potassium chloride and the like, may also be added to the uptakesolution to facilitate transformation. Similar procedures are availablefor other fungal host cells. See, e.g., U.S. Pat. No. 6,022,725.

3.3. Expression

A method of producing an amylase may comprise cultivating a host cell asdescribed above under conditions conducive to the production of theenzyme and recovering the enzyme from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof an amylase. Suitable media and media components are available fromcommercial suppliers or may be prepared according to published recipes(e.g., as described in catalogues of the American Type CultureCollection).

An enzyme secreted from the host cells can be used in a whole brothpreparation. In the present methods, the preparation of a spent wholefermentation broth of a recombinant microorganism can be achieved usingany cultivation method known in the art resulting in the expression ofan α-amylase. Fermentation may, therefore, be understood as comprisingshake flask cultivation, small- or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermenters performed in a suitable medium andunder conditions allowing the amylase to be expressed or isolated. Theterm “spent whole fermentation broth” is defined herein asunfractionated contents of fermentation material that includes culturemedium, extracellular proteins (e.g., enzymes), and cellular biomass. Itis understood that the term “spent whole fermentation broth” alsoencompasses cellular biomass that has been lysed or permeabilized usingmethods well known in the art.

An enzyme secreted from the host cells may conveniently be recoveredfrom the culture medium by well-known procedures, including separatingthe cells from the medium by centrifugation or filtration, andprecipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulfate, followed by the use of chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

The polynucleotide encoding an amylase in a vector can be operablylinked to a control sequence that is capable of providing for theexpression of the coding sequence by the host cell, i.e. the vector isan expression vector. The control sequences may be modified, for exampleby the addition of further transcriptional regulatory elements to makethe level of transcription directed by the control sequences moreresponsive to transcriptional modulators. The control sequences may inparticular comprise promoters.

Host cells may be cultured under suitable conditions that allowexpression of an amylase. Expression of the enzymes may be constitutivesuch that they are continually produced, or inducible, requiring astimulus to initiate expression. In the case of inducible expression,protein production can be initiated when required by, for example,addition of an inducer substance to the culture medium, for exampledexamethasone or IPTG or Sophorose. Polypeptides can also be producedrecombinantly in an in vitro cell-free system, such as the TNT™(Promega) rabbit reticulocyte system.

An expression host also can be cultured in the appropriate medium forthe host, under aerobic conditions. Shaking or a combination ofagitation and aeration can be provided, with production occurring at theappropriate temperature for that host, e.g., from about 25° C. to about75° C. (e.g., 30° C. to 45° C.), depending on the needs of the host andproduction of the desired variant amylase. Culturing can occur fromabout 12 to about 100 hours or greater (and any hour value therebetween, e.g., from 24 to 72 hours). Typically, the culture broth is ata pH of about 4.0 to about 8.0, again depending on the cultureconditions needed for the host relative to production of an amylase.

3.4. Identification of Amylase Activity

To evaluate the expression of an amylase in a host cell, assays canmeasure the expressed protein, corresponding mRNA, or α-amylaseactivity. For example, suitable assays include Northern blotting,reverse transcriptase polymerase chain reaction, and in situhybridization, using an appropriately labeled hybridizing probe.Suitable assays also include measuring amylase activity in a sample, forexample, by assays directly measuring reducing sugars such as glucose inthe culture media. For example, glucose concentration may be determinedusing glucose reagent kit No. 15-UV (Sigma Chemical Co.) or aninstrument, such as Technicon Autoanalyzer. α-Amylase activity also maybe measured by any known method, such as the PAHBAH or ABTS assays,described below.

3.5. Methods for Enriching and Purifying Variants Amylases

Fermentation, separation, and concentration techniques are well known inthe art and conventional methods can be used in order to prepare aconcentrated a variant α-amylase polypeptide-containing solution.

After fermentation, a fermentation broth is obtained, the microbialcells and various suspended solids, including residual raw fermentationmaterials, are removed by conventional separation techniques in order toobtain an amylase solution. Filtration, centrifugation, microfiltration,rotary vacuum drum filtration, ultrafiltration, centrifugation followedby ultrafiltration, extraction, or chromatography, or the like, aregenerally used.

It is desirable to concentrate a variant α-amylasepolypeptide-containing solution in order to optimize recovery. Use ofunconcentrated solutions requires increased incubation time in order tocollect the enriched or purified enzyme precipitate.

The enzyme containing solution is concentrated using conventionalconcentration techniques until the desired enzyme level is obtained.Concentration of the enzyme containing solution may be achieved by anyof the techniques discussed herein. Exemplary methods of enrichment andpurification include but are not limited to rotary vacuum filtrationand/or ultrafiltration.

The enzyme solution is concentrated into a concentrated enzyme solutionuntil the enzyme activity of the concentrated variant α-amylasepolypeptide-containing solution is at a desired level.

Concentration may be performed using, e.g., a precipitation agent, suchas a metal halide precipitation agent. Metal halide precipitation agentsinclude but are not limited to alkali metal chlorides, alkali metalbromides and blends of two or more of these metal halides. Exemplarymetal halides include sodium chloride, potassium chloride, sodiumbromide, potassium bromide and blends of two or more of these metalhalides. The metal halide precipitation agent, sodium chloride, can alsobe used as a preservative.

The metal halide precipitation agent is used in an amount effective toprecipitate an amylase. The selection of at least an effective amountand an optimum amount of metal halide effective to cause precipitationof the enzyme, as well as the conditions of the precipitation formaximum recovery including incubation time, pH, temperature andconcentration of enzyme, will be readily apparent to one of ordinaryskill in the art, after routine testing.

Generally, at least about 5% w/v (weight/volume) to about 25% w/v ofmetal halide is added to the concentrated enzyme solution, and usuallyat least 8% w/v. Generally, no more than about 25% w/v of metal halideis added to the concentrated enzyme solution and usually no more thanabout 20% w/v. The optimal concentration of the metal halideprecipitation agent will depend, among others, on the nature of thespecific variant α-amylase polypeptide and on its concentration in theconcentrated enzyme solution.

Another alternative way to precipitate the enzyme is to use organiccompounds. Exemplary organic compound precipitating agents include:4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid,alkyl esters of 4-hydroxybenzoic acid, and blends of two or more ofthese organic compounds. The addition of the organic compoundprecipitation agents can take place prior to, simultaneously with orsubsequent to the addition of the metal halide precipitation agent, andthe addition of both precipitation agents, organic compound and metalhalide, may be carried out sequentially or simultaneously.

Generally, the organic precipitation agents are selected from the groupconsisting of alkali metal salts of 4-hydroxybenzoic acid, such assodium or potassium salts, and linear or branched alkyl esters of4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 12carbon atoms, and blends of two or more of these organic compounds. Theorganic compound precipitation agents can be, for example, linear orbranched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl groupcontains from 1 to 10 carbon atoms, and blends of two or more of theseorganic compounds. Exemplary organic compounds are linear alkyl estersof 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 6carbon atoms, and blends of two or more of these organic compounds.Methyl esters of 4-hydroxybenzoic acid, propyl esters of4-hydroxybenzoic acid, butyl ester of 4-hydroxybenzoic acid, ethyl esterof 4-hydroxybenzoic acid and blends of two or more of these organiccompounds can also be used. Additional organic compounds also includebut are not limited to 4-hydroxybenzoic acid methyl ester (named methylPARABEN), 4-hydroxybenzoic acid propyl ester (named propyl PARABEN),which also are both amylase preservative agents. For furtherdescriptions, see, e.g., U.S. Pat. No. 5,281,526.

Addition of the organic compound precipitation agent provides theadvantage of high flexibility of the precipitation conditions withrespect to pH, temperature, variant amylase concentration, precipitationagent concentration, and time of incubation.

The organic compound precipitation agent is used in an amount effectiveto improve precipitation of the enzyme by means of the metal halideprecipitation agent. The selection of at least an effective amount andan optimum amount of organic compound precipitation agent, as well asthe conditions of the precipitation for maximum recovery includingincubation time, pH, temperature and concentration of enzyme, will bereadily apparent to one of ordinary skill in the art, in light of thepresent disclosure, after routine testing.

Generally, at least about 0.01% w/v of organic compound precipitationagent is added to the concentrated enzyme solution and usually at leastabout 0.02% w/v. Generally, no more than about 0.3% w/v of organiccompound precipitation agent is added to the concentrated enzymesolution and usually no more than about 0.2% w/v.

The concentrated polypeptide solution, containing the metal halideprecipitation agent, and the organic compound precipitation agent, canbe adjusted to a pH, which will, of necessity, depend on the enzyme tobe enriched or purified. Generally, the pH is adjusted at a level nearthe isoelectric point of the amylase. The pH can be adjusted at a pH ina range from about 2.5 pH units below the isoelectric point (pI) up toabout 2.5 pH units above the isoelectric point.

The incubation time necessary to obtain an enriched or purified enzymeprecipitate depends on the nature of the specific enzyme, theconcentration of enzyme, and the specific precipitation agent(s) and its(their) concentration. Generally, the time effective to precipitate theenzyme is between about 1 to about 30 hours; usually it does not exceedabout 25 hours. In the presence of the organic compound precipitationagent, the time of incubation can still be reduced to less about 10hours and in most cases even about 6 hours.

Generally, the temperature during incubation is between about 4° C. andabout 50° C. Usually, the method is carried out at a temperature betweenabout 10° C. and about 45° C. (e.g., between about 20° C. and about 40°C.). The optimal temperature for inducing precipitation varies accordingto the solution conditions and the enzyme or precipitation agent(s)used.

The overall recovery of enriched or purified enzyme precipitate, and theefficiency with which the process is conducted, is improved by agitatingthe solution comprising the enzyme, the added metal halide and the addedorganic compound. The agitation step is done both during addition of themetal halide and the organic compound, and during the subsequentincubation period. Suitable agitation methods include mechanicalstirring or shaking, vigorous aeration, or any similar technique.

After the incubation period, the enriched or purified enzyme is thenseparated from the dissociated pigment and other impurities andcollected by conventional separation techniques, such as filtration,centrifugation, microfiltration, rotary vacuum filtration,ultrafiltration, press filtration, cross membrane microfiltration, crossflow membrane microfiltration, or the like. Further enrichment orpurification of the enzyme precipitate can be obtained by washing theprecipitate with water. For example, the enriched or purified enzymeprecipitate is washed with water containing the metal halideprecipitation agent, or with water containing the metal halide and theorganic compound precipitation agents.

During fermentation, a variant α-amylase polypeptide accumulates in theculture broth. For the isolation, enrichment, or purification of thedesired variant α-amylase, the culture broth is centrifuged or filteredto eliminate cells, and the resulting cell-free liquid is used forenzyme enrichment or purification. In one embodiment, the cell-freebroth is subjected to salting out using ammonium sulfate at about 70%saturation; the 70% saturation-precipitation fraction is then dissolvedin a buffer and applied to a column such as a Sephadex G-100 column, andeluted to recover the enzyme-active fraction. For further enrichment orpurification, a conventional procedure such as ion exchangechromatography may be used.

Enriched or purified enzymes are useful for laundry and cleaningapplications. For example, they can be used in laundry detergents andspot removers. They can be made into a final product that is eitherliquid (solution, slurry) or solid (granular, powder).

A more specific example of enrichment or purification, is described inSumitani et al. (2000) “New type of starch-binding domain: the directrepeat motif in the C-terminal region of Bacillus sp. 195 α-amylasecontributes to starch binding and raw starch degrading,” Biochem. J.350: 477-484, and is briefly summarized here. The enzyme obtained from 4liters of a Streptomyces lividans TK24 culture supernatant was treatedwith (NH₄)₂SO₄ at 80% saturation. The precipitate was recovered bycentrifugation at 10,000×g (20 min. and 4° C.) and re-dissolved in 20 mMTris/HCl buffer (pH 7.0) containing 5 mM CaCl₂. The solubilizedprecipitate was then dialyzed against the same buffer. The dialyzedsample was then applied to a Sephacryl S-200 column, which hadpreviously been equilibrated with 20 mM Tris/HCl buffer, (pH 7.0), 5 mMCaCl₂, and eluted at a linear flow rate of 7 mL/hr with the same buffer.Fractions from the column were collected and assessed for activity asjudged by enzyme assay and SDS-PAGE. The protein was further purified asfollows. A Toyopearl HW55 column (Tosoh Bioscience, Montgomeryville,Pa.; Cat. No. 19812) was equilibrated with 20 mM Tris/HCl buffer (pH7.0) containing 5 mM CaCl₂ and 1.5 M (NH₄)₂SO₄. The enzyme was elutedwith a linear gradient of 1.5 to 0 M (NH₄)₂SO₄ in 20 mM Tris/HCL buffer,pH 7.0 containing 5 mM CaCl₂. The active fractions were collected, andthe enzyme precipitated with (NH₄)₂SO₄ at 80% saturation. Theprecipitate was recovered, re-dissolved, and dialyzed as describedabove. The dialyzed sample was then applied to a Mono Q HR5/5 column(Amersham Pharmacia; Cat. No. 17-5167-01) previously equilibrated with20 mM Tris/HCl buffer (pH 7.0) containing 5 mM CaCl₂, at a flow rate of60 mL/hour. The active fractions are collected and added to a 1.5 M(NH₄)₂SO₄ solution. The active enzyme fractions were re-chromatographedon a Toyopearl HW55 column, as before, to yield a homogeneous enzyme asdetermined by SDS-PAGE. See Sumitani et al. (2000) Biochem. J. 350:477-484, for general discussion of the method and variations thereon.

For production scale recovery, variant α-amylase polypeptides can beenriched or partially purified as generally described above by removingcells via flocculation with polymers. Alternatively, the enzyme can beenriched or purified by microfiltration followed by concentration byultrafiltration using available membranes and equipment. However, forsome applications, the enzyme does not need to be enriched or purified,and whole broth culture can be lysed and used without further treatment.The enzyme can then be processed, for example, into granules.

4. Compositions and Uses of Variant Amylases

Variants amylases are useful for a variety of industrial applications.For example, variant amylases are useful in a starch conversion process,particularly in a saccharification process of a starch that hasundergone liquefaction. The desired end-product may be any product thatmay be produced by the enzymatic conversion of the starch substrate. Forexample, the desired product may be a syrup rich in glucose and maltose,which can be used in other processes, such as the preparation of HFCS,or which can be converted into a number of other useful products, suchas ascorbic acid intermediates (e.g., gluconate; 2-keto-L-gulonic acid;5-keto-gluconate; and 2,5-diketogluconate); 1,3-propanediol; aromaticamino acids (e.g., tyrosine, phenylalanine and tryptophan); organicacids (e.g., lactate, pyruvate, succinate, isocitrate, andoxaloacetate); amino acids (e.g., serine and glycine); antibiotics;antimicrobials; enzymes; vitamins; and hormones.

The starch conversion process may be a precursor to, or simultaneouswith, a fermentation process designed to produce alcohol for fuel ordrinking (i.e., potable alcohol). One skilled in the art is aware ofvarious fermentation conditions that may be used in the production ofthese end-products. Variant amylases are also useful in compositions andmethods of food preparation. These various uses of variant amylases aredescribed in more detail below.

4.1. Preparation of Starch Substrates

Those of general skill in the art are well aware of available methodsthat may be used to prepare starch substrates for use in the processesdisclosed herein. For example, a useful starch substrate may be obtainedfrom tubers, roots, stems, legumes, cereals or whole grain. Morespecifically, the granular starch may be obtained from corn, cobs,wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca,sorghum, rice, peas, bean, banana, or potatoes. Corn contains about60-68% starch; barley contains about 55-65% starch; millet containsabout 75-80% starch; wheat contains about 60-65% starch; and polishedrice contains 70-72% starch. Specifically contemplated starch substratesare corn starch and wheat starch. The starch from a grain may be groundor whole and includes corn solids, such as kernels, bran and/or cobs.The starch may also be highly refined raw starch or feedstock fromstarch refinery processes. Various starches also are commerciallyavailable. For example, corn starch is available from Cerestar, Sigma,and Katayama Chemical Industry Co. (Japan); wheat starch is availablefrom Sigma; sweet potato starch is available from Wako Pure ChemicalIndustry Co. (Japan); and potato starch is available from NakaariChemical Pharmaceutical Co. (Japan).

The starch substrate can be a crude starch from milled whole grain,which contains non-starch fractions, e.g., germ residues and fibers.Milling may comprise either wet milling or dry milling or grinding. Inwet milling, whole grain is soaked in water or dilute acid to separatethe grain into its component parts, e.g., starch, protein, germ, oil,kernel fibers. Wet milling efficiently separates the germ and meal(i.e., starch granules and protein) and is especially suitable forproduction of syrups. In dry milling or grinding, whole kernels areground into a fine powder and often processed without fractionating thegrain into its component parts. In some cases, oils from the kernels arerecovered. Dry ground grain thus will comprise significant amounts ofnon-starch carbohydrate compounds, in addition to starch. Dry grindingof the starch substrate can be used for production of ethanol and otherbiochemicals. The starch to be processed may be a highly refined starchquality, for example, at least 90%, at least 95%, at least 97%, or atleast 99.5% pure.

4.2. Gelatinization and Liquefaction of Starch

As used herein, the term “liquefaction” or “liquefy” means a process bywhich starch is converted to less viscous and shorter chain dextrins.Generally, this process involves gelatinization of starch simultaneouslywith or followed by the addition of an α-amylase, although additionalliquefaction-inducing enzymes optionally may be added. In someembodiments, the starch substrate prepared as described above isslurried with water. The starch slurry may contain starch as a weightpercent of dry solids of about 10-55%, about 20-45%, about 30-45%, about30-40%, or about 30-35%. α-Amylase (EC 3.2.1.1) may be added to theslurry, with a metering pump, for example. The α-amylase typically usedfor this application is a thermally stable, bacterial α-amylase, such asa Geobacillus stearothermophilus α-amylase. The α-amylase is usuallysupplied, for example, at about 1500 units per kg dry matter of starch.To optimize α-amylase stability and activity, the pH of the slurrytypically is adjusted to about pH 5.5-6.5 and about 1 mM of calcium(about 40 ppm free calcium ions) can also be added. Geobacillusstearothermophilus variants or other α-amylases may require differentconditions. Bacterial α-amylase remaining in the slurry followingliquefaction may be deactivated via a number of methods, includinglowering the pH in a subsequent reaction step or by removing calciumfrom the slurry in cases where the enzyme is dependent upon calcium.

The slurry of starch plus the α-amylase may be pumped continuouslythrough a jet cooker, which is steam heated to 105° C. Gelatinizationoccurs rapidly under these conditions, and the enzymatic activity,combined with the significant shear forces, begins the hydrolysis of thestarch substrate. The residence time in the jet cooker is brief. Thepartly gelatinized starch may be passed into a series of holding tubesmaintained at 105-110° C. and held for 5-8 min. to complete thegelatinization process (“primary liquefaction”). Hydrolysis to therequired DE is completed in holding tanks at 85-95° C. or highertemperatures for about 1 to 2 hours (“secondary liquefaction”). Thesetanks may contain baffles to discourage back mixing. As used herein, theterm “minutes of secondary liquefaction” refers to the time that haselapsed from the start of secondary liquefaction to the time that theDextrose Equivalent (DE) is measured. The slurry is then allowed to coolto room temperature. This cooling step can be 30 minutes to 180 minutes,e.g. 90 minutes to 120 minutes. The liquefied starch typically is in theform of a slurry having a dry solids content (w/w) of about 10-50%;about 10-45%; about 15-40%; about 20-40%; about 25-40%; or about 25-35%.

Liquefaction with variant amylases advantageously can be conducted atlow pH, eliminating the requirement to adjust the pH to about pH5.5-6.5. Variants amylases can be used for liquefaction at a pH range of2 to 7, e.g., pH 3.0-7.5, pH 4.0-6.0, or pH 4.5-5.8. Variant amylasescan maintain liquefying activity at a temperature range of about 85°C.-95° C., e.g., 85° C., 90° C., or 95° C. For example, liquefaction canbe conducted with 800 μg an amylase in a solution of 25% DS corn starchfor 10 min at pH 5.8 and 85° C., or pH 4.5 and 95° C., for example.Liquefying activity can be assayed using any of a number of knownviscosity assays in the art.

4.3. Saccharification

The liquefied starch can be saccharified into a syrup rich in lower DP(e.g., DP1+DP2) saccharides, using variant amylases, optionally in thepresence of another enzyme(s). The exact composition of the products ofsaccharification depends on the combination of enzymes used, as well asthe type of granular starch processed. Advantageously, the syrupobtainable using the provided variant amylases may contain a weightpercent of DP2 of the total oligosaccharides in the saccharified starchexceeding 30%, e.g., 45%-65% or 55%-65%. The weight percent of (DP1+DP2)in the saccharified starch may exceed about 70%, e.g., 75%-85% or80%-85%. The present amylases also produce a relatively high yield ofglucose, e.g., DP1>20%, in the syrup product.

Whereas liquefaction is generally run as a continuous process,saccharification is often conducted as a batch process. Saccharificationtypically is most effective at temperatures of about 60-65° C. and a pHof about 4.0-4.5, e.g., pH 4.3, necessitating cooling and adjusting thepH of the liquefied starch. Saccharification may be performed, forexample, at a temperature between about 40° C., about 50° C., or about55° C. to about 60° C. or about 65° C. Saccharification is normallyconducted in stirred tanks, which may take several hours to fill orempty. Enzymes typically are added either at a fixed ratio to driedsolids as the tanks are filled or added as a single dose at thecommencement of the filling stage. A saccharification reaction to make asyrup typically is run over about 24-72 hours, for example, 24-48 hours.When a maximum or desired DE has been attained, the reaction is stoppedby heating to 85° C. for 5 min., for example. Further incubation willresult in a lower DE, eventually to about 90 DE, as accumulated glucosere-polymerizes to isomaltose and/or other reversion products via anenzymatic reversion reaction and/or with the approach of thermodynamicequilibrium. When using an amylase, saccharification optimally isconducted at a temperature range of about 30° C. to about 75° C., e.g.,45° C.-75° C. or 47° C.-74° C. The saccharifying may be conducted over apH range of about pH 3 to about pH 7, e.g., pH 3.0-pH 7.5, pH 3.5-pH5.5, pH 3.5, pH 3.8, or pH 4.5.

An amylase may be added to the slurry in the form of a composition.Amylase can be added to a slurry of a granular starch substrate in anamount of about 0.6-10 ppm ds, e.g., 2 ppm ds. An amylase can be addedas a whole broth, clarified, enriched, partially purified, or purifiedenzyme. The specific activity of the amylase may be about 300 U/mg ofenzyme, for example, measured with the PAHBAH assay. The amylase alsocan be added as a whole broth product.

An amylase may be added to the slurry as an isolated enzyme solution.For example, an amylase can be added in the form of a cultured cellmaterial produced by host cells expressing an amylase. An amylase mayalso be secreted by a host cell into the reaction medium during thefermentation or SSF process, such that the enzyme is providedcontinuously into the reaction. The host cell producing and secretingamylase may also express an additional enzyme, such as a glucoamylase.For example, U.S. Pat. No. 5,422,267 discloses the use of a glucoamylasein yeast for production of alcoholic beverages. For example, a hostcell, e.g., Trichoderma reesei or Aspergillus niger, may be engineeredto co-express an amylase and a glucoamylase, e.g., HgGA, TrGA, or a TrGAvariant, during saccharification. The host cell can be geneticallymodified so as not to express its endogenous glucoamylase and/or otherenzymes, proteins or other materials. The host cell can be engineered toexpress a broad spectrum of various saccharolytic enzymes. For example,the recombinant yeast host cell can comprise nucleic acids encoding aglucoamylase, an alpha-glucosidase, an enzyme that utilizes pentosesugar, an α-amylase, a pullulanse, an isoamylase, and/or anisopullulanase. See, e.g., WO 2011/153516 A2.

4.4. Isomerization

The soluble starch hydrolysate produced by treatment with amylase can beconverted into high fructose starch-based syrup (HFSS), such as highfructose corn syrup (HFCS). This conversion can be achieved using aglucose isomerase, particularly a glucose isomerase immobilized on asolid support. The pH is increased to about 6.0 to about 8.0, e.g., pH7.5 (depending on the isomerase), and Ca²⁺ is removed by ion exchange.Suitable isomerases include Sweetzyme®, IT (Novozymes A/S); G-Zyme®IMGI, and G-Zyme® G993, Ketomax®, G-Zyme® G993, G-Zyme® G993 liquid, andGenSweet® IGI. Following isomerization, the mixture typically containsabout 40-45% fructose, e.g., 42% fructose.

4.5. Fermentation

The soluble starch hydrolysate, particularly a glucose rich syrup, canbe fermented by contacting the starch hydrolysate with a fermentingorganism typically at a temperature around 32° C., such as from 30° C.to 35° C. for alcohol-producing yeast. The temperature and pH of thefermentation will depend upon the fermenting organism. EOF productsinclude metabolites, such as citric acid, lactic acid, succinic acid,monosodium glutamate, gluconic acid, sodium gluconate, calciumgluconate, potassium gluconate, itaconic acid and other carboxylicacids, glucono delta-lactone, sodium erythorbate, lysine and other aminoacids, omega 3 fatty acid, butanol, isoprene, 1,3-propanediol and otherbiomaterials.

Ethanologenic microorganisms include yeast, such as Saccharomycescerevisiae and bacteria, e.g., Zymomonas moblis, expressing alcoholdehydrogenase and pyruvate decarboxylase. The ethanologenicmicroorganism can express xylose reductase and xylitol dehydrogenase,which convert xylose to xylulose Improved strains of ethanologenicmicroorganisms, which can withstand higher temperatures, for example,are known in the art and can be used. See Liu et al. (2011) Sheng WuGong Cheng Xue Bao 27(7): 1049-56. Commercial sources of yeast includeETHANOL RED® (LeSaffre); Thermosacc® (Lallemand); RED STAR® (Red Star);FERMIOL® (DSM Specialties); and SUPERSTART® (Alltech). Microorganismsthat produce other metabolites, such as citric acid and lactic acid, byfermentation are also known in the art. See, e.g., Papagianni (2007)“Advances in citric acid fermentation by Aspergillus niger: biochemicalaspects, membrane transport and modeling,” Biotechnol. Adv. 25(3):244-63; John et al. (2009) “Direct lactic acid fermentation: focus onsimultaneous saccharification and lactic acid production,” Biotechnol.Adv. 27(2): 145-52.

The saccharification and fermentation processes may be carried out as anSSF process. Fermentation may comprise subsequent enrichment,purification, and recovery of ethanol, for example. During thefermentation, the ethanol content of the broth or “beer” may reach about8-18% v/v, e.g., 14-15% v/v. The broth may be distilled to produceenriched, e.g., 96% pure, solutions of ethanol. Further, CO₂ generatedby fermentation may be collected with a CO₂ scrubber, compressed, andmarketed for other uses, e.g., carbonating beverage or dry iceproduction. Solid waste from the fermentation process may be used asprotein-rich products, e.g., livestock feed.

As mentioned above, an SSF process can be conducted with fungal cellsthat express and secrete amylase continuously throughout SSF. The fungalcells expressing amylase also can be the fermenting microorganism, e.g.,an ethanologenic microorganism. Ethanol production thus can be carriedout using a fungal cell that expresses sufficient amylase so that lessor no enzyme has to be added exogenously. The fungal host cell can befrom an appropriately engineered fungal strain. Fungal host cells thatexpress and secrete other enzymes, in addition to amylase, also can beused. Such cells may express glucoamylase and/or a pullulanase, phytase,alpha-glucosidase, isoamylase, beta-amylase cellulase, xylanase, otherhemicellulases, protease, beta-glucosidase, pectinase, esterase, redoxenzymes, transferase, or other enzyme.

A variation on this process is a “fed-batch fermentation” system, wherethe substrate is added in increments as the fermentation progresses.Fed-batch systems are useful when catabolite repression may inhibit themetabolism of the cells and where it is desirable to have limitedamounts of substrate in the medium. The actual substrate concentrationin fed-batch systems is estimated by the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gases,such as CO₂. Batch and fed-batch fermentations are common and well knownin the art.

Continuous fermentation is an open system where a defined fermentationmedium is added continuously to a bioreactor, and an equal amount ofconditioned medium is removed simultaneously for processing. Continuousfermentation generally maintains the cultures at a constant high densitywhere cells are primarily in log phase growth. Continuous fermentationpermits modulation of cell growth and/or product concentration. Forexample, a limiting nutrient such as the carbon source or nitrogensource is maintained at a fixed rate and all other parameters areallowed to moderate. Because growth is maintained at a steady state,cell loss due to medium being drawn off should be balanced against thecell growth rate in the fermentation. Methods of optimizing continuousfermentation processes and maximizing the rate of product formation arewell known in the art of industrial microbiology.

4.6. Compositions Comprising Variants Amylases

Variant amylases may be combined with a glucoamylase (EC 3.2.1.3), e.g.,a Trichoderma glucoamylase or variant thereof. An exemplary glucoamylaseis Trichoderma reesei glucoamylase (TrGA) and variants thereof thatpossess superior specific activity and thermal stability. See U.S.Published Applications Nos. 2006/0094080, 2007/0004018, and 2007/0015266(Danisco US Inc.). Suitable variants of TrGA include those withglucoamylase activity and at least 80%, at least 90%, or at least 95%sequence identity to wild-type TrGA. Variant amylases advantageouslyincrease the yield of glucose produced in a saccharification processcatalyzed by TrGA.

Alternatively, the glucoamylase may be another glucoamylase derived fromplants (including algae), fungi, or bacteria. For example, theglucoamylases may be Aspergillus niger G1 or G2 glucoamylase or itsvariants (e.g., Boel et al. (1984) EMBO J. 3: 1097-1102; WO 92/00381; WO00/04136 (Novo Nordisk A/S)); and A. awamori glucoamylase (e.g., WO84/02921 (Cetus Corp.)). Other contemplated Aspergillus glucoamylaseinclude variants with enhanced thermal stability, e.g., G137A and G139A(Chen et al. (1996) Prot. Eng. 9: 499-505); D257E and D293E/Q (Chen etal. (1995) Prot. Eng. 8: 575-582); N182 (Chen et al. (1994) Biochem. J.301: 275-281); A246C (Fierobe et al. (1996) Biochemistry, 35:8698-8704); and variants with Pro residues in positions A435 and S436(Li et al. (1997) Protein Eng. 10: 1199-1204). Other contemplatedglucoamylases include Talaromyces glucoamylases, in particular derivedfrom T. emersonii (e.g., WO 99/28448 (Novo Nordisk A/S), T. leycettanus(e.g., U.S. Pat. No. RE 32,153 (CPC International, Inc.)), T. duponti,or T. thermophilus (e.g., U.S. Pat. No. 4,587,215). Contemplatedbacterial glucoamylases include glucoamylases from the genusClostridium, in particular C. thermoamylolyticum (e.g., EP 135,138 (CPCInternational, Inc.) and C. thermohydrosulfuricum (e.g., WO 86/01831(Michigan Biotechnology Institute)). Suitable glucoamylases include theglucoamylases derived from Aspergillus oryzae, such as a glucoamylaseshown in SEQ ID NO:2 in WO 00/04136 (Novo Nordisk A/S). Also suitableare commercial glucoamylases, such as AMG 200L; AMG 300 L; SAN™ SUPERand AMG™ E (Novozymes); OPTIDEX® 300 and OPTIDEX L-400 (Danisco USInc.); AMIGASE™ and AMIGASE™ PLUS (DSM); G-ZYME® G900 (EnzymeBio-Systems); and G-ZYME® G990 ZR (A. niger glucoamylase with a lowprotease content). Still other suitable glucoamylases includeAspergillus fumigatus glucoamylase, Talaromyces glucoamylase, Thielaviaglucoamylase, Trametes glucoamylase, Thermomyces glucoamylase, Atheliaglucoamylase, or Humicola glucoamylase (e.g., HgGA). Glucoamylasestypically are added in an amount of about 0.1-2 glucoamylase units(GAU)/g ds, e.g., about 0.16 GAU/g ds, 0.23 GAU/g ds, or 0.33 GAU/g ds.

Other suitable enzymes that can be used with amylase include a phytase,protease, pullulanase, β-amylase, isoamylase, a different α-amylase,alpha-glucosidase, cellulase, xylanase, other hemicellulases,beta-glucosidase, transferase, pectinase, lipase, cutinase, esterase,redox enzymes, or a combination thereof. For example, a debranchingenzyme, such as an isoamylase (EC 3.2.1.68), may be added in effectiveamounts well known to the person skilled in the art. A pullulanase (EC3.2.1.41), e.g., Promozyme®, is also suitable. Pullulanase typically isadded at 100 U/kg ds. Further suitable enzymes include proteases, suchas fungal and bacterial proteases. Fungal proteases include thoseobtained from Aspergillus, such as A. niger, A. awamori, A. oryzae;Mucor (e.g., M. miehei); Rhizopus; and Trichoderma.

β-Amylases (EC 3.2.1.2) are exo-acting maltogenic amylases, whichcatalyze the hydrolysis of 1,4-α-glucosidic linkages into amylopectinand related glucose polymers, thereby releasing maltose. β-Amylases havebeen isolated from various plants and microorganisms. See Fogarty et al.(1979) in PROGRESS IN INDUSTRIAL MICROBIOLOGY, Vol. 15, pp. 112-115.These β-Amylases have optimum temperatures in the range from 40° C. to65° C. and optimum pH in the range from about 4.5 to about 7.0.Contemplated β-amylases include, but are not limited to, β-amylases frombarley Spezyme® BBA 1500, Spezyme® DBA, Optimalt™ ME, Optimalt™ BBA(Danisco US Inc.); and Novozym™ WBA (Novozymes A/S).

Compositions comprising the present amylases may be aqueous ornon-aqueous formulations, granules, powders, gels, slurries, pastes,etc., which may further comprise any one or more of the additionalenzymes listed, herein, along with buffers, salts, preservatives, water,co-solvents, surfactants, and the like. Such compositions may work incombination with endogenous enzymes or other ingredients already presentin a slurry, water bath, washing machine, food or drink product, etc,for example, endogenous plant (including algal) enzymes, residualenzymes from a prior processing step, and the like.

5. Compositions and Methods for Baking and Food Preparation

The present invention also relates to a “food composition,” includingbut not limited to a food product, animal feed and/or food/feedadditives, comprising an amylase, and methods for preparing such a foodcomposition comprising mixing variant amylase with one or more foodingredients, or uses thereof.

Furthermore, the present invention relates to the use of an amylase inthe preparation of a food composition, wherein the food composition isbaked subsequent to the addition of the polypeptide of the invention. Asused herein the term “baking composition” means any composition and/oradditive prepared in the process of providing a baked food product,including but not limited to bakers flour, a dough, a baking additiveand/or a baked product. The food composition or additive may be liquidor solid.

As used herein, the term “flour” means milled or ground cereal grain.The term “flour” also may mean Sago or tuber products that have beenground or mashed. In some embodiments, flour may also contain componentsin addition to the milled or mashed cereal or plant matter. An exampleof an additional component, although not intended to be limiting, is aleavening agent. Cereal grains include wheat, oat, rye, and barley.Tuber products include tapioca flour, cassava flour, and custard powder.The term “flour” also includes ground corn flour, maize-meal, riceflour, whole-meal flour, self-rising flour, tapioca flour, cassavaflour, ground rice, enriched flower, and custard powder.

For the commercial and home use of flour for baking and food production,it is important to maintain an appropriate level of α-amylase activityin the flour. A level of activity that is too high may result in aproduct that is sticky and/or doughy and therefore unmarketable. Flourwith insufficient α-amylase activity may not contain enough sugar forproper yeast function, resulting in dry, crumbly bread, or bakedproducts. Accordingly, an amylase, by itself or in combination withanother α-amylase(s), may be added to the flour to augment the level ofendogenous α-amylase activity in flour.

An amylase can further be added alone or in a combination with otheramylases to prevent or retard staling, i.e., crumb firming of bakedproducts. The amount of anti-staling amylase will typically be in therange of 0.01-10 mg of enzyme protein per kg of flour, e.g., 0.5 mg/kgds. Additional anti-staling amylases that can be used in combinationwith an amylase include an endo-amylase, e.g., a bacterial endo-amylasefrom Bacillus. The additional amylase can be another maltogenicα-amylase (EC 3.2.1.133), e.g., from Bacillus. Novamyl® is an exemplarymaltogenic α-amylase from B. stearothermophilus strain NCIB 11837 and isdescribed in Christophersen et al. (1997) Starch 50: 39-45. Otherexamples of anti-staling endo-amylases include bacterial α-amylasesderived from Bacillus, such as B. licheniformis or B. amyloliquefaciens.The anti-staling amylase may be an exo-amylase, such as β-amylase, e.g.,from plant sources, such as soy bean, or from microbial sources, such asBacillus.

The baking composition comprising an amylase further can comprise aphospholipase or enzyme with phospholipase activity. An enzyme withphospholipase activity has an activity that can be measured in LipaseUnits (LU). The phospholipase may have A₁ or A₂ activity to remove fattyacid from the phospholipids, forming a lysophospholipid. It may or maynot have lipase activity, i.e., activity on triglyceride substrates. Thephospholipase typically has a temperature optimum in the range of 30-90°C., e.g., 30-70° C. The added phospholipases can be of animal origin,for example, from pancreas, e.g., bovine or porcine pancreas, snakevenom or bee venom. Alternatively, the phospholipase may be of microbialorigin, e.g., from filamentous fungi, yeast or bacteria, for example.

The phospholipase is added in an amount that improves the softness ofthe bread during the initial period after baking, particularly the first24 hours. The amount of phospholipase will typically be in the range of0.01-10 mg of enzyme protein per kg of flour, e.g., 0.1-5 mg/kg. Thatis, phospholipase activity generally will be in the range of 20-1000LU/kg of flour, where a Lipase Unit is defined as the amount of enzymerequired to release 1 μmol butyric acid per minute at 30° C., pH 7.0,with gum arabic as emulsifier and tributyrin as substrate.

Compositions of dough generally comprise wheat meal or wheat flourand/or other types of meal, flour or starch such as corn flour,cornstarch, rye meal, rye flour, oat flour, oatmeal, soy flour, sorghummeal, sorghum flour, potato meal, potato flour or potato starch. Thedough may be fresh, frozen or par-baked. The dough can be a leaveneddough or a dough to be subjected to leavening. The dough may be leavenedin various ways, such as by adding chemical leavening agents, e.g.,sodium bicarbonate or by adding a leaven, i.e., fermenting dough. Doughalso may be leavened by adding a suitable yeast culture, such as aculture of Saccharomyces cerevisiae (baker's yeast), e.g., acommercially available strain of S. cerevisiae.

The dough may also comprise other conventional dough ingredients, e.g.,proteins, such as milk powder, gluten, and soy; eggs (e.g., whole eggs,egg yolks or egg whites); an oxidant, such as ascorbic acid, potassiumbromate, potassium iodate, azodicarbonamide (ADA) or ammoniumpersulfate; an amino acid such as L-cysteine; a sugar; or a salt, suchas sodium chloride, calcium acetate, sodium sulfate or calcium sulfate.The dough further may comprise fat, e.g., triglyceride, such asgranulated fat or shortening. The dough further may comprise anemulsifier such as mono- or diglycerides, diacetyl tartaric acid estersof mono- or diglycerides, sugar esters of fatty acids, polyglycerolesters of fatty acids, lactic acid esters of monoglycerides, acetic acidesters of monoglycerides, polyoxyethylene stearates, or lysolecithin. Inparticular, the dough can be made without addition of emulsifiers.

The dough product may be any processed dough product, including fried,deep fried, roasted, baked, steamed and boiled doughs, such as steamedbread and rice cakes. In one embodiment, the food product is a bakeryproduct. Typical bakery (baked) products include bread—such as loaves,rolls, buns, bagels, pizza bases etc. pastry, pretzels, tortillas,cakes, cookies, biscuits, crackers etc.

Optionally, an additional enzyme may be used together with theanti-staling amylase and the phospholipase. The additional enzyme may bea second amylase, such as an amyloglucosidase, a β-amylase, acyclodextrin glucanotransferase, or the additional enzyme may be apeptidase, in particular an exopeptidase, a transglutaminase, a lipase,a cellulase, a xylanase, a protease, a protein disulfide isomerase,e.g., a protein disulfide isomerase as disclosed in WO 95/00636, forexample, a glycosyltransferase, a branching enzyme (1,4-α-glucanbranching enzyme), a 4-α-glucanotransferase (dextringlycosyltransferase) or an oxidoreductase, e.g., a peroxidase, alaccase, a glucose oxidase, a pyranose oxidase, a lipooxygenase, anL-amino acid oxidase or a carbohydrate oxidase. The additional enzyme(s)may be of any origin, including mammalian and plant, and particularly ofmicrobial (bacterial, yeast or fungal) origin and may be obtained bytechniques conventionally used in the art.

The xylanase is typically of microbial origin, e.g., derived from abacterium or fungus, such as a strain of Aspergillus. Xylanases includePentopan® and Novozym 384®, for example, which are commerciallyavailable xylanase preparations produced from Trichoderma reesei. Theamyloglucosidase may be an A. niger amyloglucosidase (such as AMG®).Other useful amylase products include Grindamyl® A 1000 or A 5000(Grindsted Products, Denmark) and Amylase® H or Amylase® P (DSM). Theglucose oxidase may be a fungal glucose oxidase, in particular anAspergillus niger glucose oxidase (such as Gluzyme®). An exemplaryprotease is Neutrase®.

The process may be used for any kind of baked product prepared fromdough, either of a soft or a crisp character, either of a white, lightor dark type. Examples are bread, particularly white, whole-meal or ryebread, typically in the form of loaves or rolls, such as, but notlimited to, French baguette-type bread, pita bread, tortillas, cakes,pancakes, biscuits, cookies, pie crusts, crisp bread, steamed bread,pizza and the like.

An amylase may be used in a pre-mix, comprising flour together with ananti-staling amylase, a phospholipase, and/or a phospholipid. Thepre-mix may contain other dough-improving and/or bread-improvingadditives, e.g., any of the additives, including enzymes, mentionedabove. An amylase can be a component of an enzyme preparation comprisingan anti-staling amylase and a phospholipase, for use as a bakingadditive.

The enzyme preparation is optionally in the form of a granulate oragglomerated powder. The preparation can have a narrow particle sizedistribution with more than 95% (by weight) of the particles in therange from 25 to 500 μm. Granulates and agglomerated powders may beprepared by conventional methods, e.g., by spraying an amylase onto acarrier in a fluid-bed granulator. The carrier may consist ofparticulate cores having a suitable particle size. The carrier may besoluble or insoluble, e.g., a salt (such as NaCl or sodium sulfate), asugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol),starch, rice, corn grits, or soy.

Enveloped particles, i.e., α-amylase particles, can comprise an amylase.To prepare enveloped α-amylase particles, the enzyme is contacted with afood grade lipid in sufficient quantity to suspend all of the α-amylaseparticles. Food grade lipids, as used herein, may be any naturallyorganic compound that is insoluble in water but is soluble in non-polarorganic solvents such as hydrocarbon or diethyl ether. Suitable foodgrade lipids include, but are not limited to, triglycerides either inthe form of fats or oils that are either saturated or unsaturated.Examples of fatty acids and combinations thereof which make up thesaturated triglycerides include, but are not limited to, butyric(derived from milk fat), palmitic (derived from animal and plant fat),and/or stearic (derived from animal and plant fat). Examples of fattyacids and combinations thereof which make up the unsaturatedtriglycerides include, but are not limited to, palmitoleic (derived fromanimal and plant fat), oleic (derived from animal and plant fat),linoleic (derived from plant oils), and/or linolenic (derived fromlinseed oil). Other suitable food grade lipids include, but are notlimited to, monoglycerides and diglycerides derived from thetriglycerides discussed above, phospholipids and glycolipids.

The food grade lipid, particularly in the liquid form, is contacted witha powdered form of the α-amylase particles in such a fashion that thelipid material covers at least a portion of the surface of at least amajority, e.g., 100% of the α-amylase particles. Thus, each α-amylaseparticle is individually enveloped in a lipid. For example, all orsubstantially all of the α-amylase particles are provided with a thin,continuous, enveloping film of lipid. This can be accomplished by firstpouring a quantity of lipid into a container, and then slurrying theα-amylase particles so that the lipid thoroughly wets the surface ofeach α-amylase particle. After a short period of stirring, the envelopedα-amylase particles, carrying a substantial amount of the lipids ontheir surfaces, are recovered. The thickness of the coating so appliedto the particles of α-amylase can be controlled by selection of the typeof lipid used and by repeating the operation in order to build up athicker film, when desired.

The storing, handling and incorporation of the loaded delivery vehiclecan be accomplished by means of a packaged mix. The packaged mix cancomprise the enveloped α-amylase. However, the packaged mix may furthercontain additional ingredients as required by the manufacturer or baker.After the enveloped α-amylase has been incorporated into the dough, thebaker continues through the normal production process for that product.

The advantages of enveloping the α-amylase particles are two-fold.First, the food grade lipid protects the enzyme from thermaldenaturation during the baking process for those enzymes that are heatlabile. Consequently, while the α-amylase is stabilized and protectedduring the proving and baking stages, it is released from the protectivecoating in the final baked good product, where it hydrolyzes theglucosidic linkages in polyglucans. The loaded delivery vehicle alsoprovides a sustained release of the active enzyme into the baked good.That is, following the baking process, active α-amylase is continuallyreleased from the protective coating at a rate that counteracts, andtherefore reduces the rate of, staling mechanisms.

In general, the amount of lipid applied to the α-amylase particles canvary from a few percent of the total weight of the α-amylase to manytimes that weight, depending upon the nature of the lipid, the manner inwhich it is applied to the α-amylase particles, the composition of thedough mixture to be treated, and the severity of the dough-mixingoperation involved.

The loaded delivery vehicle, i.e., the lipid-enveloped enzyme, is addedto the ingredients used to prepare a baked good in an effective amountto extend the shelf-life of the baked good. The baker computes theamount of enveloped α-amylase, prepared as discussed above, that will berequired to achieve the desired anti-staling effect. The amount of theenveloped α-amylase required is calculated based on the concentration ofenzyme enveloped and on the proportion of α-amylase to flour specified.A wide range of concentrations has been found to be effective, although,as has been discussed, observable improvements in anti-staling do notcorrespond linearly with the α-amylase concentration, but above certainminimal levels, large increases in α-amylase concentration producelittle additional improvement. The α-amylase concentration actually usedin a particular bakery production could be much higher than the minimumnecessary to provide the baker with some insurance against inadvertentunder-measurement errors by the baker. The lower limit of enzymeconcentration is determined by the minimum anti-staling effect the bakerwishes to achieve.

A method of preparing a baked good may comprise: a) preparinglipid-coated α-amylase particles, where substantially all of theα-amylase particles are coated; b) mixing a dough containing flour; c)adding the lipid-coated α-amylase to the dough before the mixing iscomplete and terminating the mixing before the lipid coating is removedfrom the α-amylase; d) proofing the dough; and e) baking the dough toprovide the baked good, where the α-amylase is inactive during themixing, proofing and baking stages and is active in the baked good.

The enveloped α-amylase can be added to the dough during the mix cycle,e.g., near the end of the mix cycle. The enveloped α-amylase is added ata point in the mixing stage that allows sufficient distribution of theenveloped α-amylase throughout the dough; however, the mixing stage isterminated before the protective coating becomes stripped from theα-amylase particle(s). Depending on the type and volume of dough, andmixer action and speed, anywhere from one to six minutes or more mightbe required to mix the enveloped α-amylase into the dough, but two tofour minutes is average. Thus, several variables may determine theprecise procedure. First, the quantity of enveloped α-amylase shouldhave a total volume sufficient to allow the enveloped α-amylase to bespread throughout the dough mix. If the preparation of envelopedα-amylase is highly concentrated, additional oil may need to be added tothe pre-mix before the enveloped α-amylase is added to the dough.Recipes and production processes may require specific modifications;however, good results generally can be achieved when 25% of the oilspecified in a bread dough formula is held out of the dough and is usedas a carrier for a concentrated enveloped α-amylase when added near theend of the mix cycle. In bread or other baked goods, particularly thosehaving a low fat content, e.g., French-style breads, an envelopedα-amylase mixture of approximately 1% of the dry flour weight issufficient to admix the enveloped α-amylase properly with the dough. Therange of suitable percentages is wide and depends on the formula,finished product, and production methodology requirements of theindividual baker. Second, the enveloped α-amylase suspension should beadded to the mix with sufficient time for complete mixture into thedough, but not for such a time that excessive mechanical action stripsthe protective lipid coating from the enveloped α-amylase particles.

In a further aspect of the invention, the food composition is an oil,meat, lard, composition comprising an amylase. In this context the term“oil/meat/lard” composition” means any composition, based on, made fromand/or containing oil, meat or lard, respectively. Another aspect theinvention relates to a method of preparing an oil or meat or lardcomposition and/or additive comprising an amylase, comprising mixing thepolypeptide of the invention with a oil/meat/lard composition and/oradditive ingredients.

In a further aspect of the invention, the food composition is an animalfeed composition, animal feed additive and/or pet food comprising anamylase and variants thereof. The present invention further relates to amethod for preparing such an animal feed composition, animal feedadditive composition and/or pet food comprising mixing an amylase andvariants thereof with one or more animal feed ingredients and/or animalfeed additive ingredients and/or pet food ingredients. Furthermore, thepresent invention relates to the use of an amylase in the preparation ofan animal feed composition and/or animal feed additive compositionand/or pet food.

The term “animal” includes all non-ruminant and ruminant animals. In aparticular embodiment, the animal is a non-ruminant animal, such as ahorse and a mono-gastric animal.

Examples of mono-gastric animals include, but are not limited to, pigsand swine, such as piglets, growing pigs, sows; poultry such as turkeys,ducks, chicken, broiler chicks, layers; fish such as salmon, trout,tilapia, catfish and carps; and crustaceans such as shrimps and prawns.In a further embodiment the animal is a ruminant animal including, butnot limited to, cattle, young calves, goats, sheep, giraffes, bison,moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas,antelope, pronghorn and nilgai.

In the present context, it is intended that the term “pet food” isunderstood to mean a food for a household animal such as, but notlimited to dogs, cats, gerbils, hamsters, chinchillas, fancy rats,guinea pigs; avian pets, such as canaries, parakeets, and parrots;reptile pets, such as turtles, lizards and snakes; and aquatic pets,such as tropical fish and frogs.

The terms “animal feed composition,” “feedstuff” and “fodder” are usedinterchangeably and may comprise one or more feed materials selectedfrom the group comprising a) cereals, such as small grains (e.g., wheat,barley, rye, oats and combinations thereof) and/or large grains such asmaize or sorghum; b) by products from cereals, such as corn gluten meal,Distillers Dried Grain Solubles (DDGS) (particularly corn basedDistillers Dried Grain Solubles (cDDGS), wheat bran, wheat middlings,wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citruspulp; c) protein obtained from sources such as soya, sunflower, peanut,lupin, peas, fava beans, cotton, canola, fish meal, dried plasmaprotein, meat and bone meal, potato protein, whey, copra, sesame; d)oils and fats obtained from vegetable and animal sources; e) mineralsand vitamins

6. Textile Desizing Compositions and Use

Also contemplated are compositions and methods of treating fabrics(e.g., to desize a textile) using an an amylase. Fabric-treating methodsare well known in the art (see, e.g., U.S. Pat. No. 6,077,316). Forexample, the feel and appearance of a fabric can be improved by a methodcomprising contacting the fabric with an an amylase in a solution. Thefabric can be treated with the solution under pressure.

An amylase can be applied during or after the weaving of a textile, orduring the desizing stage, or one or more additional fabric processingsteps. During the weaving of textiles, the threads are exposed toconsiderable mechanical strain. Prior to weaving on mechanical looms,warp yarns are often coated with sizing starch or starch derivatives toincrease their tensile strength and to prevent breaking. An an amylasecan be applied during or after the weaving to remove these sizing starchor starch derivatives. After weaving, an an amylase can be used toremove the size coating before further processing the fabric to ensure ahomogeneous and wash-proof result.

An amylase can be used alone or with other desizing chemical reagentsand/or desizing enzymes to desize fabrics, including cotton-containingfabrics, as detergent additives, e.g., in aqueous compositions. An anamylase also can be used in compositions and methods for producing astonewashed look on indigo-dyed denim fabric and garments. For themanufacture of clothes, the fabric can be cut and sewn into clothes orgarments, which are afterwards finished. In particular, for themanufacture of denim jeans, different enzymatic finishing methods havebeen developed. The finishing of denim garment normally is initiatedwith an enzymatic desizing step, during which garments are subjected tothe action of amylolytic enzymes to provide softness to the fabric andmake the cotton more accessible to the subsequent enzymatic finishingsteps. An an amylase can be used in methods of finishing denim garments(e.g., a “bio-stoning process”), enzymatic desizing and providingsoftness to fabrics, and/or finishing process.

7. Cleaning Compositions

An aspect of the present compositions and methods is a cleaningcomposition that includes an amylase as a component. An amylasepolypeptide can be used as a component in detergent compositions forhand washing, laundry washing, dishwashing, and other hard-surfacecleaning.

7.1. Overview

Preferably, an amylase is incorporated into detergents at or near aconcentration conventionally used for amylase in detergents. Forexample, an amylase polypeptide may be added in amount corresponding to0.00001-1 mg (calculated as pure enzyme protein) of amylase per liter ofwash/dishwash liquor. Exemplary formulations are provided herein, asexemplified by the following:

An amylase polypeptide may be a component of a detergent composition, asthe only enzyme or with other enzymes including other amylolyticenzymes. As such, it may be included in the detergent composition in theform of a non-dusting granulate, a stabilized liquid, or a protectedenzyme. Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated bymethods known in the art. Examples of waxy coating materials arepoly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molarweights of 1,000 to 20,000; ethoxylated nonylphenols having from 16 to50 ethylene oxide units; ethoxylated fatty alcohols in which the alcoholcontains from 12 to 20 carbon atoms and in which there are 15 to 80ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in, forexample, GB 1483591. Liquid enzyme preparations may, for instance, bestabilized by adding a polyol such as propylene glycol, a sugar or sugaralcohol, lactic acid or boric acid according to established methods.Other enzyme stabilizers are known in the art. Protected enzymes may beprepared according to the method disclosed in for example EP 238 216.Polyols have long been recognized as stabilizers of proteins, as well asimproving protein solubility.

The detergent composition may be in any useful form, e.g., as powders,granules, pastes, or liquid. A liquid detergent may be aqueous,typically containing up to about 70% of water and 0% to about 30% oforganic solvent. It may also be in the form of a compact gel typecontaining only about 30% water.

The detergent composition comprises one or more surfactants, each ofwhich may be anionic, nonionic, cationic, or zwitterionic. The detergentwill usually contain 0% to about 50% of anionic surfactant, such aslinear alkylbenzenesulfonate (LAS); α-olefinsulfonate (AOS); alkylsulfate (fatty alcohol sulfate) (AS); alcohol ethoxysulfate (AEOS orAES); secondary alkanesulfonates (SAS); α-sulfo fatty acid methylesters; alkyl- or alkenylsuccinic acid; or soap. The composition mayalso contain 0% to about 40% of nonionic surfactant such as alcoholethoxylate (AEO or AE), carboxylated alcohol ethoxylates, nonylphenolethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylatedfatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxyalkyl fatty acid amide (as described for example in WO 92/06154).

The detergent composition may additionally comprise one or more otherenzymes, such as proteases, another amylolytic enzyme, cutinase, lipase,cellulase, pectate lyase, perhydrolase, xylanase, peroxidase, and/orlaccase in any combination.

The detergent may contain about 1% to about 65% of a detergent builderor complexing agent such as zeolite, diphosphate, triphosphate,phosphonate, citrate, nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates orlayered silicates (e.g., SKS-6 from Hoechst). The detergent may also beunbuilt, i.e. essentially free of detergent builder. The enzymes can beused in any composition compatible with the stability of the enzyme.Enzymes generally can be protected against deleterious components byknown forms of encapsulation, for example, by granulation orsequestration in hydro gels. Enzymes, and specifically amylases, eitherwith or without starch binding domains, can be used in a variety ofcompositions including laundry and dishwashing applications, surfacecleaners, as well as in compositions for ethanol production from starchor biomass.

The detergent may comprise one or more polymers. Examples includecarboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system, which may comprise a H₂O₂source such as perborate or percarbonate, which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine(TAED) or nonanoyloxybenzenesulfonate (NOBS). Alternatively, thebleaching system may comprise peroxyacids (e.g., the amide, imide, orsulfone type peroxyacids). The bleaching system can also be an enzymaticbleaching system, for example, perhydrolase, such as that described inInternational PCT Application WO 2005/056783.

The enzymes of the detergent composition may be stabilized usingconventional stabilizing agents, e.g., a polyol such as propylene glycolor glycerol; a sugar or sugar alcohol; lactic acid; boric acid or aboric acid derivative such as, e.g., an aromatic borate ester; and thecomposition may be formulated as described in, e.g., WO 92/19709 and WO92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, tarnish inhibiters, opticalbrighteners, or perfumes.

The pH (measured in aqueous solution at use concentration) is usuallyneutral or alkaline, e.g., pH about 7.0 to about 11.0.

Particular forms of detergent compositions for inclusion of the presentα-amylase are described, below.

7.2. Heavy Duty Liquid (HDL) Laundry Detergent Composition

Exemplary HDL laundry detergent compositions includes a detersivesurfactant (10%-40% wt/wt), including an anionic detersive surfactant(selected from a group of linear or branched or random chain,substituted or unsubstituted alkyl sulphates, alkyl sulphonates, alkylalkoxylated sulphate, alkyl phosphates, alkyl phosphonates, alkylcarboxylates, and/or mixtures thereof), and optionally non-ionicsurfactant (selected from a group of linear or branched or random chain,substituted or unsubstituted alkyl alkoxylated alcohol, for example aC₈-C₁₈ alkyl ethoxylated alcohol and/or C₆-C₁₂ alkyl phenolalkoxylates), wherein the weight ratio of anionic detersive surfactant(with a hydrophilic index (HIc) of from 6.0 to 9) to non-ionic detersivesurfactant is greater than 1:1. Suitable detersive surfactants alsoinclude cationic detersive surfactants (selected from a group of alkylpyridinium compounds, alkyl quarternary ammonium compounds, alkylquarternary phosphonium compounds, alkyl ternary sulphonium compounds,and/or mixtures thereof); zwitterionic and/or amphoteric detersivesurfactants (selected from a group of alkanolamine sulpho-betaines);ampholytic surfactants; semi-polar non-ionic surfactants and mixturesthereof.

The composition may optionally include, a surfactancy boosting polymerconsisting of amphiphilic alkoxylated grease cleaning polymers (selectedfrom a group of alkoxylated polymers having branched hydrophilic andhydrophobic properties, such as alkoxylated polyalkylenimines in therange of 0.05 wt %-10 wt %) and/or random graft polymers (typicallycomprising of hydrophilic backbone comprising monomers selected from thegroup consisting of: unsaturated C₁-C₆ carboxylic acids, ethers,alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleicanhydride, saturated polyalcohols such as glycerol, and mixturesthereof; and hydrophobic side chain(s) selected from the groupconsisting of: C₄-C₂₅ alkyl group, polypropylene, polybutylene, vinylester of a saturated C₁-C₆ mono-carboxylic acid, C₁-C₆ alkyl ester ofacrylic or methacrylic acid, and mixtures thereof.

The composition may include additional polymers such as soil releasepolymers (include anionically end-capped polyesters, for example SRP1,polymers comprising at least one monomer unit selected from saccharide,dicarboxylic acid, polyol and combinations thereof, in random or blockconfiguration, ethylene terephthalate-based polymers and co-polymersthereof in random or block configuration, for example Repel-o-tex SF,SF-2 and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300and SRN325, Marloquest SL), anti-redeposition polymers (0.1 wt % to 10wt %, include carboxylate polymers, such as polymers comprising at leastone monomer selected from acrylic acid, maleic acid (or maleicanhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid,citraconic acid, methylenemalonic acid, and any mixture thereof,vinylpyrrolidone homopolymer, and/or polyethylene glycol, molecularweight in the range of from 500 to 100,000 Da); cellulosic polymer(including those selected from alkyl cellulose, alkyl alkoxyalkylcellulose, carboxyalkyl cellulose, alkyl carboxyalkyl cellulose examplesof which include carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixuresthereof) and polymeric carboxylate (such as maleate/acrylate randomcopolymer or polyacrylate homopolymer).

The composition may further include saturated or unsaturated fatty acid,preferably saturated or unsaturated C₁₂-C₂₄ fatty acid (0 wt % to 10 wt%); deposition aids (examples for which include polysaccharides,preferably cellulosic polymers, poly diallyl dimethyl ammonium halides(DADMAC), and co-polymers of DAD MAC with vinyl pyrrolidone,acrylamides, imidazoles, imidazolinium halides, and mixtures thereof, inrandom or block configuration, cationic guar gum, cationic cellulosesuch as cationic hydoxyethyl cellulose, cationic starch, cationicpolyacylamides, and mixtures thereof.

The composition may further include dye transfer inhibiting agents,examples of which include manganese phthalocyanine, peroxidases,polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers ofN-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinylimidazoles and/or mixtures thereof; chelating agents, examplesof which include ethylene-diamine-tetraacetic acid (EDTA), diethylenetriamine penta methylene phosphonic acid (DTPMP), hydroxy-ethanediphosphonic acid (HEDP), ethylenediamine N,N′-disuccinic acid (EDDS),methyl glycine diacetic acid (MGDA), diethylene triamine penta aceticacid (DTPA), propylene diamine tetracetic acid (PDT A),2-hydroxypyridine-N-oxide (HPNO), or methyl glycine diacetic acid(MGDA), glutamic acid N,N-diacetic acid (N,N-dicarboxymethyl glutamicacid tetrasodium salt (GLDA), nitrilotriacetic acid (NTA),4,5-dihydroxy-m-benzenedisulfonic acid, citric acid and any saltsthereof, N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiaceticacid (HEIDA), dihydroxyethylglycine (DHEG),ethylenediaminetetrapropionic acid (EDTP), and derivatives thereof.

The composition preferably included enzymes (generally about 0.01 wt %active enzyme to 0.03 wt % active enzyme) selected from proteases,amylases, lipases, cellulases, choline oxidases, peroxidases/oxidases,pectate lyases, mannanases, cutinases, laccases, phospholipases,lysophospholipases, acyltransferases, perhydrolases, arylesterases, andany mixture thereof. The composition may include an enzyme stabilizer(examples of which include polyols such as propylene glycol or glycerol,sugar or sugar alcohol, lactic acid, reversible protease inhibitor,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronicacid).

The composition optionally include silicone or fatty-acid based sudssuppressors; heuing dyes, calcium and magnesium cations, visualsignaling ingredients, anti-foam (0.001 wt % to about 4.0 wt %), and/orstructurant/thickener (0.01 wt % to 5 wt %, selected from the groupconsisting of diglycerides and triglycerides, ethylene glycoldistearate, microcrystalline cellulose, cellulose based materials,microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixturesthereof).

The composition can be any liquid form, for example a liquid or gelform, or any combination thereof. The composition may be in any unitdose form, for example a pouch.

7.3. Heavy Duty Dry/Solid (HDD) Laundry Detergent Composition

Exemplary HDD laundry detergent compositions includes a detersivesurfactant, including anionic detersive surfactants (e.g., linear orbranched or random chain, substituted or unsubstituted alkyl sulphates,alkyl sulphonates, alkyl alkoxylated sulphate, alkyl phosphates, alkylphosphonates, alkyl carboxylates and/or mixtures thereof), non-ionicdetersive surfactant (e.g., linear or branched or random chain,substituted or unsubstituted C₈-C₁₈ alkyl ethoxylates, and/or C₆-C₁₂alkyl phenol alkoxylates), cationic detersive surfactants (e.g., alkylpyridinium compounds, alkyl quaternary ammonium compounds, alkylquaternary phosphonium compounds, alkyl ternary sulphonium compounds,and mixtures thereof), zwitterionic and/or amphoteric detersivesurfactants (e.g., alkanolamine sulpho-betaines), ampholyticsurfactants, semi-polar non-ionic surfactants, and mixtures thereof;builders including phosphate free builders (for example zeolite buildersexamples which include zeolite A, zeolite X, zeolite P and zeolite MAPin the range of 0 wt % to less than 10 wt %), phosphate builders (forexample sodium tri-polyphosphate in the range of 0 wt % to less than 10wt %), citric acid, citrate salts and nitrilotriacetic acid, silicatesalt (e.g., sodium or potassium silicate or sodium meta-silicate in therange of 0 wt % to less than 10 wt %, or layered silicate (SKS-6));carbonate salt (e.g., sodium carbonate and/or sodium bicarbonate in therange of 0 wt % to less than 80 wt %); and bleaching agents includingphotobleaches (e.g., sulfonated zinc phthalocyanines, sulfonatedaluminum phthalocyanines, xanthenes dyes, and mixtures thereof)hydrophobic or hydrophilic bleach activators (e.g., dodecanoyloxybenzene sulfonate, decanoyl oxybenzene sulfonate, decanoyl oxybenzoicacid or salts thereof, 3,5,5-trimethy hexanoyl oxybenzene sulfonate,tetraacetyl ethylene diamine-TAED, nonanoyloxybenzene sulfonate-NOBS,nitrile quats, and mixtures thereof), sources of hydrogen peroxide(e.g., inorganic perhydrate salts examples of which include mono ortetra hydrate sodium salt of perborate, percarbonate, persulfate,perphosphate, or persilicate), preformed hydrophilic and/or hydrophobicperacids (e.g., percarboxylic acids and salts, percarbonic acids andsalts, perimidic acids and salts, peroxymonosulfuric acids and salts,and mixtures thereof), and/or bleach catalysts (e.g., imine bleachboosters (examples of which include iminium cations and polyions),iminium zwitterions, modified amines, modified amine oxides, N-sulphonylimines, N-phosphonyl imines, N-acyl imines, thiadiazole dioxides,perfluoroimines, cyclic sugar ketones, and mixtures thereof, andmetal-containing bleach catalysts (e.g., copper, iron, titanium,ruthenium, tungsten, molybdenum, or manganese cations along with anauxiliary metal cations such as zinc or aluminum and a sequestrate suchas ethylenediaminetetraacetic acid,ethylenediaminetetra(methylenephosphonic acid), and water-soluble saltsthereof).

The composition preferably includes enzymes, e.g., proteases, amylases,lipases, cellulases, choline oxidases, peroxidases/oxidases, pectatelyases, mannanases, cutinases, laccases, phospholipases,lysophospholipases, acyltransferase, perhydrolase, arylesterase, and anymixture thereof.

The composition may optionally include additional detergent ingredientsincluding perfume microcapsules, starch encapsulated perfume accord,hueing agents, additional polymers, including fabric integrity andcationic polymers, dye-lock ingredients, fabric-softening agents,brighteners (for example C.I. Fluorescent brighteners), flocculatingagents, chelating agents, alkoxylated polyamines, fabric depositionaids, and/or cyclodextrin.

7.4. Automatic Dishwashing (ADW) Detergent Composition

Exemplary ADW detergent composition includes non-ionic surfactants,including ethoxylated non-ionic surfactants, alcohol alkoxylatedsurfactants, epoxy-capped poly(oxyalkylated) alcohols, or amine oxidesurfactants present in amounts from 0 to 10% by weight; builders in therange of 5-60% including phosphate builders (e.g., mono-phosphates,di-phosphates, tri-polyphosphates, other oligomeric-poylphosphates,sodium tripolyphosphate-STPP) and phosphate-free builders (e.g., aminoacid-based compounds including methyl-glycine-diacetic acid (MGDA) andsalts and derivatives thereof, glutamic-N,N-diacetic acid (GLDA) andsalts and derivatives thereof, iminodisuccinic acid (IDS) and salts andderivatives thereof, carboxy methyl inulin and salts and derivativesthereof, nitrilotriacetic acid (NTA), diethylene triamine penta aceticacid (DTPA), B-alaninediacetic acid (B-ADA) and their salts,homopolymers and copolymers of poly-carboxylic acids and their partiallyor completely neutralized salts, monomeric polycarboxylic acids andhydroxycarboxylic acids and their salts in the range of 0.5% to 50% byweight; sulfonated/carboxylated polymers in the range of about 0.1% toabout 50% by weight to provide dimensional stability; drying aids in therange of about 0.1% to about 10% by weight (e.g., polyesters, especiallyanionic polyesters, optionally together with further monomers with 3 to6 functionalities—typically acid, alcohol or ester functionalities whichare conducive to polycondensation, polycarbonate-, polyurethane- and/orpolyurea-polyorganosiloxane compounds or precursor compounds, thereof,particularly of the reactive cyclic carbonate and urea type); silicatesin the range from about 1% to about 20% by weight (including sodium orpotassium silicates for example sodium disilicate, sodium meta-silicateand crystalline phyllosilicates); inorganic bleach (e.g., perhydratesalts such as perborate, percarbonate, perphosphate, persulfate andpersilicate salts) and organic bleach (e.g., organic peroxyacids,including diacyl and tetraacylperoxides, especially diperoxydodecanediocacid, diperoxytetradecanedioc acid, and diperoxyhexadecanedioc acid);bleach activators (i.e., organic peracid precursors in the range fromabout 0.1% to about 10% by weight); bleach catalysts (e.g., manganesetriazacyclononane and related complexes, Co, Cu, Mn, and Febispyridylamine and related complexes, and pentamine acetate cobalt(III)and related complexes); metal care agents in the range from about 0.1%to 5% by weight (e.g., benzatriazoles, metal salts and complexes, and/orsilicates); enzymes in the range from about 0.01 to 5.0 mg of activeenzyme per gram of automatic dishwashing detergent composition (e.g.,proteases, amylases, lipases, cellulases, choline oxidases,peroxidases/oxidases, pectate lyases, mannanases, cutinases, laccases,phospholipases, lysophospholipases, acyltransferase, perhydrolase,arylesterase, and mixtures thereof); and enzyme stabilizer components(e.g., oligosaccharides, polysaccharides, and inorganic divalent metalsalts).

7.5. Additional Detergent Compositions

Additional exemplary detergent formulations to which the present amylasecan be added are described, below, in the numbered paragraphs.

1) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising linear alkylbenzenesulfonate(calculated as acid) about 7% to about 12%; alcohol ethoxysulfate (e.g.,C₁₂₋₁₈ alcohol, 1-2 ethylene oxide (EO)) or alkyl sulfate (e.g., C₁₆₋₁₈)about 1% to about 4%; alcohol ethoxylate (e.g., C₁₄₋₁₅ alcohol, 7 EO)about 5% to about 9%; sodium carbonate (e.g., Na₂CO₃) about 14% to about20%; soluble silicate (e.g., Na₂O, 2SiO₂) about 2 to about 6%; zeolite(e.g., NaAlSiO₄) about 15% to about 22%; sodium sulfate (e.g., Na₂SO₄)0% to about 6%; sodium citrate/citric acid (e.g., C₆H₅Na₃O₇/C₆H₈O₇)about 0% to about 15%; sodium perborate (e.g., NaBO₃H₂O) about 11% toabout 18%; TAED about 2% to about 6%; carboxymethylcellulose (CMC) and0% to about 2%; polymers (e.g., maleic/acrylic acid, copolymer, PVP,PEG) 0-3%; enzymes (calculated as pure enzyme) 0.0001-0.1% protein; andminor ingredients (e.g., suds suppressors, perfumes, optical brightener,photobleach) 0-5%.

2) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising linear alkylbenzenesulfonate(calculated as acid) about 6% to about 11%; alcohol ethoxysulfate (e.g.,C₁₂₋₁₈ alcohol, 1-2 EO) or alkyl sulfate (e.g., C₁₆₋₁₈) about 1% toabout 3%; alcohol ethoxylate (e.g., C₁₄₋₁₅ alcohol, 7 EO) about 5% toabout 9%; sodium carbonate (e.g., Na₂CO₃) about 15% to about 21%;soluble silicate (e.g., Na₂O, 2SiO₂) about 1% to about 4%; zeolite(e.g., NaAlSiO₄) about 24% to about 34%; sodium sulfate (e.g., Na₂SO₄)about 4% to about 10%; sodium citrate/citric acid (e.g.,C₆H₅Na₃O₇/C₆H₈O₇) 0% to about 15%; carboxymethylcellulose (CMC) 0% toabout 2%; polymers (e.g., maleic/acrylic acid copolymer, PVP, PEG) 1-6%;enzymes (calculated as pure enzyme protein) 0.0001-0.1%; minoringredients (e.g., suds suppressors, perfume) 0-5%.

3) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising linear alkylbenzenesulfonate(calculated as acid) about 5% to about 9%; alcohol ethoxylate (e.g.,C₁₂₋₁₅ alcohol, 7 EO) about 7% to about 14%; Soap as fatty acid (e.g.,C₁₆₋₂₂ fatty acid) about 1 to about 3%; sodium carbonate (as Na₂CO₃)about 10% to about 17%; soluble silicate (e.g., Na₂O, 2SiO₂) about 3% toabout 9%; zeolite (as NaAlSiO₄) about 23% to about 33%; sodium sulfate(e.g., Na₂SO₄) 0% to about 4%; sodium perborate (e.g., NaBO₃H₂O) about8% to about 16%; TAED about 2% to about 8%; phosphonate (e.g., EDTMPA)0% to about 1%; carboxymethylcellulose (CMC) 0% to about 2%; polymers(e.g., maleic/acrylic acid copolymer, PVP, PEG) 0-3%; enzymes(calculated as pure enzyme protein) 0.0001-0.1%; minor ingredients(e.g., suds suppressors, perfume, optical brightener) 0-5%.

4) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising linear alkylbenzenesulfonate(calculated as acid) about 8% to about 12%; alcohol ethoxylate (e.g.,C₁₂₋₁₅ alcohol, 7 EO) about 10% to about 25%; sodium carbonate (asNa₂CO₃) about 14% to about 22%; soluble silicate (e.g., Na₂O, 2SiO₂)about 1% to about 5%; zeolite (e.g., NaAlSiO₄) about 25% to about 35%;sodium sulfate (e.g., Na₂SO₄) 0% to about 10%; carboxymethylcellulose(CMC) 0% to about 2%; polymers (e.g., maleic/acrylic acid copolymer,PVP, PEG) 1-3%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%;and minor ingredients (e.g., suds suppressors, perfume) 0-5%.

5) An aqueous liquid detergent composition comprising linearalkylbenzenesulfonate (calculated as acid) about 15% to about 21%;alcohol ethoxylate (e.g., C₁₂₋₁₅ alcohol, 7 EO or C₁₂₋₁₅ alcohol, 5 EO)about 12% to about 18%; soap as fatty acid (e.g., oleic acid) about 3%to about 13%; alkenylsuccinic acid (C₁₂₋₁₄) 0% to about 13%;aminoethanol about 8% to about 18%; citric acid about 2% to about 8%;phosphonate 0% to about 3%; polymers (e.g., PVP, PEG) 0% to about 3%;borate (e.g., B₄O₇) 0% to about 2%; ethanol 0% to about 3%; propyleneglycol about 8% to about 14%; enzymes (calculated as pure enzymeprotein) 0.0001-0.1%; and minor ingredients (e.g., dispersants, sudssuppressors, perfume, optical brightener) 0-5%.

6) An aqueous structured liquid detergent composition comprising linearalkylbenzenesulfonate (calculated as acid) about 15% to about 21%;alcohol ethoxylate (e.g., C₁₂₋₁₅ alcohol, 7 EO, or C₁₂₋₁₅ alcohol, 5 EO)3-9%; soap as fatty acid (e.g., oleic acid) about 3% to about 10%;zeolite (as NaAlSiO₄) about 14% to about 22%; potassium citrate about 9%to about 18%; borate (e.g., B₄O₇) 0% to about 2%; carboxymethylcellulose(CMC) 0% to about 2%; polymers (e.g., PEG, PVP) 0% to about 3%;anchoring polymers such as, e.g., lauryl methacrylate/acrylic acidcopolymer; molar ratio 25:1, MW 3800) 0% to about 3%; glycerol 0% toabout 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; andminor ingredients (e.g., dispersants, suds suppressors, perfume, opticalbrighteners) 0-5%.

7) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising fatty alcohol sulfate about 5% toabout 10%; ethoxylated fatty acid monoethanolamide about 3% to about 9%;soap as fatty acid 0-3%; sodium carbonate (e.g., Na₂CO₃) about 5% toabout 10%; Soluble silicate (e.g., Na₂O, 2SiO₂) about 1% to about 4%;zeolite (e.g., NaAlSiO₄) about 20% to about 40%; Sodium sulfate (e.g.,Na₂SO₄) about 2% to about 8%; sodium perborate (e.g., NaBO₃H₂O) about12% to about 18%; TAED about 2% to about 7%; polymers (e.g.,maleic/acrylic acid copolymer, PEG) about 1% to about 5%; enzymes(calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients(e.g., optical brightener, suds suppressors, perfume) 0-5%.

8) A detergent composition formulated as a granulate comprising linearalkylbenzenesulfonate (calculated as acid) about 8% to about 14%;ethoxylated fatty acid monoethanolamide about 5% to about 11%; soap asfatty acid 0% to about 3%; sodium carbonate (e.g., Na₂CO₃) about 4% toabout 10%; soluble silicate (Na₂O, 2SiO₂) about 1% to about 4%; zeolite(e.g., NaAlSiO₄) about 30% to about 50%; sodium sulfate (e.g., Na₂SO₄)about 3% to about 11%; sodium citrate (e.g., C₆H₅Na₃O₇) about 5% toabout 12%; polymers (e.g., PVP, maleic/acrylic acid copolymer, PEG)about 1% to about 5%; enzymes (calculated as pure enzyme protein)0.0001-0.1%; and minor ingredients (e.g., suds suppressors, perfume)0-5%.

9) A detergent composition formulated as a granulate comprising linearalkylbenzenesulfonate (calculated as acid) about 6% to about 12%;nonionic surfactant about 1% to about 4%; soap as fatty acid about 2% toabout 6%; sodium carbonate (e.g., Na₂CO₃) about 14% to about 22%;zeolite (e.g., NaAlSiO₄) about 18% to about 32%; sodium sulfate (e.g.,Na₂SO₄) about 5% to about 20%; sodium citrate (e.g., C₆H₅Na₃O₇) about 3%to about 8%; sodium perborate (e.g., NaBO₃H₂O) about 4% to about 9%;bleach activator (e.g., NOBS or TAED) about 1% to about 5%;carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g.,polycarboxylate or PEG) about 1% to about 5%; enzymes (calculated aspure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., opticalbrightener, perfume) 0-5%.

10) An aqueous liquid detergent composition comprising linearalkylbenzenesulfonate (calculated as acid) about 15% to about 23%;alcohol ethoxysulfate (e.g., C₁₂₋₁₅ alcohol, 2-3 EO) about 8% to about15%; alcohol ethoxylate (e.g., C₁₂₋₁₅ alcohol, 7 EO, or C₁₂₋₁₅ alcohol,5 EO) about 3% to about 9%; soap as fatty acid (e.g., lauric acid) 0% toabout 3%; aminoethanol about 1% to about 5%; sodium citrate about 5% toabout 10%; hydrotrope (e.g., sodium toluensulfonate) about 2% to about6%; borate (e.g., B₄O₇) 0% to about 2%; carboxymethylcellulose 0% toabout 1%; ethanol about 1% to about 3%; propylene glycol about 2% toabout 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; andminor ingredients (e.g., polymers, dispersants, perfume, opticalbrighteners) 0-5%.

11) An aqueous liquid detergent composition comprising linearalkylbenzenesulfonate (calculated as acid) about 20% to about 32%;alcohol ethoxylate (e.g., C₁₂₋₁₅ alcohol, 7 EO, or C₁₂₋₁₅ alcohol, 5 EO)6-12%; aminoethanol about 2% to about 6%; citric acid about 8% to about14%; borate (e.g., B₄O₇) about 1% to about 3%; polymer (e.g.,maleic/acrylic acid copolymer, anchoring polymer such as, e.g., laurylmethacrylate/acrylic acid copolymer) 0% to about 3%; glycerol about 3%to about 8%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%;and minor ingredients (e.g., hydrotropes, dispersants, perfume, opticalbrighteners) 0-5%.

12) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising anionic surfactant (linearalkylbenzenesulfonate, alkyl sulfate, α-olefinsulfonate, α-sulfo fattyacid methyl esters, alkanesulfonates, soap) about 25% to about 40%;nonionic surfactant (e.g., alcohol ethoxylate) about 1% to about 10%;sodium carbonate (e.g., Na₂CO₃) about 8% to about 25%; soluble silicates(e.g., Na₂O, 2SiO₂) about 5% to about 15%; sodium sulfate (e.g., Na₂SO₄)0% to about 5%; zeolite (NaAlSiO₄) about 15% to about 28%; sodiumperborate (e.g., NaBO₃.4H₂O) 0% to about 20%; bleach activator (TAED orNOBS) about 0% to about 5%; enzymes (calculated as pure enzyme protein)0.0001-0.1%; minor ingredients (e.g., perfume, optical brighteners)0-3%.

13) Detergent compositions as described in compositions 1)-12) supra,wherein all or part of the linear alkylbenzenesulfonate is replaced by(C₁₂-C₁₈) alkyl sulfate.

14) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising (C₁₂-C₁₈) alkyl sulfate about 9%to about 15%; alcohol ethoxylate about 3% to about 6%; polyhydroxy alkylfatty acid amide about 1% to about 5%; zeolite (e.g., NaAlSiO₄) about10% to about 20%; layered disilicate (e.g., SK56 from Hoechst) about 10%to about 20%; sodium carbonate (e.g., Na₂CO₃) about 3% to about 12%;soluble silicate (e.g., Na₂O, 2SiO₂) 0% to about 6%; sodium citrateabout 4% to about 8%; sodium percarbonate about 13% to about 22%; TAEDabout 3% to about 8%; polymers (e.g., polycarboxylates and PVP) 0% toabout 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; andminor ingredients (e.g., optical brightener, photobleach, perfume, sudssuppressors) 0-5%.

15) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising (C₁₂-C₁₈) alkyl sulfate about 4%to about 8%; alcohol ethoxylate about 11% to about 15%; soap about 1% toabout 4%; zeolite MAP or zeolite A about 35% to about 45%; sodiumcarbonate (as Na₂CO₃) about 2% to about 8%; soluble silicate (e.g.,Na₂O, 2SiO₂) 0% to about 4%; sodium percarbonate about 13% to about 22%;TAED 1-8%; carboxymethylcellulose (CMC) 0% to about 3%; polymers (e.g.,polycarboxylates and PVP) 0% to about 3%; enzymes (calculated as pureenzyme protein) 0.0001-0.1%; and minor ingredients (e.g., opticalbrightener, phosphonate, perfume) 0-3%.

16) Detergent formulations as described in 1)-15) supra, which contain astabilized or encapsulated peracid, either as an additional component oras a substitute for already specified bleach systems.

17) Detergent compositions as described supra in 1), 3), 7), 9), and12), wherein perborate is replaced by percarbonate.

18) Detergent compositions as described supra in 1), 3), 7), 9), 12),14), and 15), which additionally contain a manganese catalyst. Themanganese catalyst for example is one of the compounds described in“Efficient manganese catalysts for low-temperature bleaching,” Nature369: 637-639 (1994).

19) Detergent composition formulated as a non-aqueous detergent liquidcomprising a liquid nonionic surfactant such as, e.g., linearalkoxylated primary alcohol, a builder system (e.g., phosphate), anenzyme(s), and alkali. The detergent may also comprise anionicsurfactant and/or a bleach system.

As above, the present amylase polypeptide may be incorporated at aconcentration conventionally employed in detergents. It is at presentcontemplated that, in the detergent composition, the enzyme may be addedin an amount corresponding to 0.00001-1.0 mg (calculated as pure enzymeprotein) of amylase polypeptide per liter of wash liquor.

The detergent composition may also contain other conventional detergentingredients, e.g., deflocculant material, filler material, foamdepressors, anti-corrosion agents, soil-suspending agents, sequesteringagents, anti-soil redeposition agents, dehydrating agents, dyes,bactericides, fluorescers, thickeners, and perfumes.

The detergent composition may be formulated as a hand (manual) ormachine (automatic) laundry detergent composition, including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for manual or automatic dishwashingoperations.

Any of the cleaning compositions described, herein, may include anynumber of additional enzymes. In general the enzyme(s) should becompatible with the selected detergent, (e.g., with respect topH-optimum, compatibility with other enzymatic and non-enzymaticingredients, and the like), and the enzyme(s) should be present ineffective amounts. The following enzymes are provided as examples.

Proteases:

Suitable proteases include those of animal, vegetable or microbialorigin. Chemically modified or protein engineered mutants are included,as well as naturally processed proteins. The protease may be a serineprotease or a metalloprotease, an alkaline microbial protease, atrypsin-like protease, or a chymotrypsin-like protease. Examples ofalkaline proteases are subtilisins, especially those derived fromBacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309,subtilisin 147, and subtilisin 168 (see, e.g., WO 89/06279). Examples oftrypsin-like proteases are trypsin (e.g., of porcine or bovine origin),and Fusarium proteases (see, e.g., WO 89/06270 and WO 94/25583).Examples of useful proteases also include but are not limited to thevariants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO98/34946. Commercially available protease enzymes include but are notlimited to: ALCALASE®, SAVINASE®, PRIMASE™, DURALASE™, ESPERASE®,KANNASE™ and BLAZE™ (Novo Nordisk A/S and Novozymes A/S); MAXATASE®,MAXACAL™, MAXAPEM™, PROPERASE®, PURAFECT®, PURAFECT OXP™ FN2™, and FN3™(Danisco US Inc.). Other exemplary proteases include NprE from Bacillusamyloliquifaciens and ASP from Cellulomonas sp. strain 69B4.

Lipases:

Suitable lipases include those of bacterial or fungal origin. Chemicallymodified, proteolytically modified, or protein engineered mutants areincluded. Examples of useful lipases include but are not limited tolipases from Humicola (synonym Thermomyces), e.g., from H. lanuginosa(T. lanuginosus) (see e.g., EP 258068 and EP 305216), from H. insolens(see e.g., WO 96/13580); a Pseudomonas lipase (e.g., from P. alcaligenesor P. pseudoalcaligenes; see, e.g., EP 218 272), P. cepacia (see e.g.,EP 331 376), P. stutzeri (see e.g., GB 1,372,034), P. fluorescens,Pseudomonas sp. strain SD 705 (see e.g., WO 95/06720 and WO 96/27002),P. wisconsinensis (see e.g., WO 96/12012); a Bacillus lipase (e.g., fromB. subtilis; see e.g., Dartois et al. Biochemica et Biophysica Acta,1131: 253-360 (1993)), B. stearothermophilus (see e.g., JP 64/744992),or B. pumilus (see e.g., WO 91/16422). Additional lipase variantscontemplated for use in the formulations include those described forexample in: WO 92/05249, WO 94/01541, WO 95/35381, WO 96/00292, WO95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO97/07202, EP 407225, and EP 260105. Some commercially available lipaseenzymes include LIPOLASE® and LIPOLASE ULTRA™ (Novo Nordisk A/S andNovozymes A/S).

Polyesterases:

Suitable polyesterases can be included in the composition, such as thosedescribed in, for example, WO 01/34899, WO 01/14629, and U.S. Pat. No.6,933,140.

Amylases: The compositions can be combined with other amylases, such asnon-production enhanced amylase. These can include commerciallyavailable amylases, such as but not limited to STAINZYME®, NATALASE®,DURAMYL®, TERMAMYL®, FUNGAMYL® and BAN™ (Novo Nordisk A/S and NovozymesA/S); RAPIDASE®, POWERASE®, and PURASTAR® (from Danisco US Inc.).

Cellulases:

Cellulases can be added to the compositions. Suitable cellulases includethose of bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Suitable cellulases include cellulasesfrom the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia,Acremonium, e.g., the fungal cellulases produced from Humicola insolens,Myceliophthora thermophila and Fusarium oxysporum disclosed for examplein U.S. Pat. Nos. 4,435,307; 5,648,263; 5,691,178; 5,776,757; and WO89/09259. Exemplary cellulases contemplated for use are those havingcolor care benefit for the textile. Examples of such cellulases arecellulases described in for example EP 0495257, EP 0531372, WO 96/11262,WO 96/29397, and WO 98/08940. Other examples are cellulase variants,such as those described in WO 94/07998; WO 98/12307; WO 95/24471;PCT/DK98/00299; EP 531315; U.S. Pat. Nos. 5,457,046; 5,686,593; and5,763,254. Commercially available cellulases include CELLUZYME® andCAREZYME® (Novo Nordisk A/S and Novozymes A/S); CLAZINASE® and PURADAXHA® (Danisco US Inc.); and KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases:

Suitable peroxidases/oxidases contemplated for use in the compositionsinclude those of plant, bacterial or fungal origin. Chemically modifiedor protein engineered mutants are included. Examples of usefulperoxidases include peroxidases from Coprinus, e.g., from C. cinereus,and variants thereof as those described in WO 93/24618, WO 95/10602, andWO 98/15257. Commercially available peroxidases include for exampleGUARDZYME™ (Novo Nordisk A/S and Novozymes A/S).

The detergent composition can also comprise 2,6-β-D-fructan hydrolase,which is effective for removal/cleaning of biofilm present on householdand/or industrial textile/laundry.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,i.e. a separate additive or a combined additive, can be formulated e.g.,as a granulate, a liquid, a slurry, and the like. Exemplary detergentadditive formulations include but are not limited to granulates, inparticular non-dusting granulates, liquids, in particular stabilizedliquids or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (e.g., polyethyleneglycol, PEG) with mean molar weightsof 1,000 to 20,000; ethoxylated nonylphenols having from 16 to 50ethylene oxide units; ethoxylated fatty alcohols in which the alcoholcontains from 12 to 20 carbon atoms and in which there are 15 to 80ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in, forexample, GB 1483591. Liquid enzyme preparations may, for instance, bestabilized by adding a polyol such as propylene glycol, a sugar or sugaralcohol, lactic acid or boric acid according to established methods.Protected enzymes may be prepared according to the method disclosed inEP 238,216.

The detergent composition may be in any convenient form, e.g., a bar, atablet, a powder, a granule, a paste, or a liquid. A liquid detergentmay be aqueous, typically containing up to about 70% water, and 0% toabout 30% organic solvent. Compact detergent gels containing about 30%or less water are also contemplated. The detergent composition canoptionally comprise one or more surfactants, which may be non-ionic,including semi-polar and/or anionic and/or cationic and/or zwitterionic.The surfactants can be present in a wide range, from about 0.1% to about60% by weight.

When included therein the detergent will typically contain from about 1%to about 40% of an anionic surfactant, such as linearalkylbenzenesulfonate, α-olefinsulfonate, alkyl sulfate (fatty alcoholsulfate), alcohol ethoxysulfate, secondary alkanesulfonate, α-sulfofatty acid methyl ester, alkyl- or alkenylsuccinic acid, or soap.

When included therein, the detergent will usually contain from about0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl-N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0% to about 65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, triphosphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid, alkyl- or alkenylsuccinic acid, soluble silicates or layeredsilicates (e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Exemplary polymersinclude carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA),poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylatese.g., polyacrylates, maleic/acrylic acid copolymers), and laurylmethacrylate/acrylic acid copolymers.

The enzyme(s) of the detergent composition may be stabilized usingconventional stabilizing agents, e.g., as polyol (e.g., propylene glycolor glycerol), a sugar or sugar alcohol, lactic acid, boric acid, or aboric acid derivative (e.g., an aromatic borate ester), or a phenylboronic acid derivative (e.g., 4-formylphenyl boronic acid). Thecomposition may be formulated as described in WO 92/19709 and WO92/19708.

It is contemplated that in the detergent compositions, in particular theenzyme variants, may be added in an amount corresponding to about 0.01to about 100 mg of enzyme protein per liter of wash liquor (e.g., about0.05 to about 5.0 mg of enzyme protein per liter of wash liquor or 0.1to about 1.0 mg of enzyme protein per liter of wash liquor).

Yet additional exemplary detergent formulations to which the presentamylase can be added (or is in some cases identified as a component) arelisted in the following Tables:

HDL Detergent Composition Ingredient wt % Enzyme (s) (Protease +Lipase + Amylase) 3 Linear alkyl benzene sulphonic acid (HLAS) 10 C12-14alkyl ethoxylated alcohol having an 2 average degree of ethoxylation of9 (AE9) C12-14 alkyl ethoxylated sulphonic acid having an 23 averagedegree of ethoxylation of 3 (HAES) C16-17 alkyl mid chain branched alkylsulphate 4 Amine oxide 1 C12-18 fatty acid 2 PE20 polymer 3 Polyethyleneimine polymer 3 Chelant 1.4 FW A 15 Brightener 0.4 p-glycol (solvent) 8DEG (solvent) 0.5 Ethanol 3 Monoethanolamine 6 Water 26 NaOH 0.3 Perfume1 Silicone suds suppressor 0.06 Violet DD dye 0.01 Other dyes 0.03Hydrogenated castor oil (structurant/thickener) 0.1 Mica 0.2 Calciumformate 0.1 Sodium formate 0.2 Miscellaneous to 100

HDD Detergent Compositions Compo- Compo- Compo- Compo- sition sitionsition sition Ingredient A B C D Enzyme 0.8 wt % 0.8 wt % 0.8 wt % 0.8wt % (Lipase + other enzymes) Linear alkyl 9 wt % 9 wt % 12 wt % 8 wt %benzene sulphonate Alkyl 3 wt % 2 wt % 1 wt % 2 wt % ethoxylatedsulphate having an average degree of ethoxylation of from 0.5 to 3Cationic 0.5 wt % 0.5 wt % 0.5 wt % 0.5 wt % detersive surfactant Sodium55 wt % 55 wt % 55 wt % 55 wt % sulphate Sodium 8 wt % 10 wt % 5 wt % 8wt % carbonate Glycerol 9 wt % 12 wt % 8 wt % 10 wt % carbonate Oxaziri-0.005 wt % 0.005 wt % 0.005 wt % 0.005 wt % diniuym- based bleachcatalyst Sodium 3 wt % 0 wt % 3 wt % 0 wt % silicate Carboxylate 2 wt %2 wt % 2 wt % 2 wt % polymer Brightener 0.02 wt % 0.02 wt % 0.02 wt %0.02 wt % Cellulosic 0.3 wt % 0.3 wt % 0.3 wt % 0.3 wt % polymer Misc &to 100 wt % to 100 wt % to 100 wt % to 100 wt % Moisture

HDD Detergent Compositions 1 2 3 4 5 6 (wt (wt (wt (wt (wt (wtIngredient %) %) %) %) %) %) Sodium linear 10.3 10.7 14 17 12.2 8.3alkylbenzenesulfonate with average aliphatic chain length C11-12 Sodiumlauryl sulfate 0 3.5 0 1.4 1.2 0 Sodium C12-14 alcohol 0 0 0.8 0 0 3ethoxy-3-sulfate C13-15 oxo alcohol 1.57 0 0 0 1.2 0 ethoxylate withaverage 7 moles of ethoxylation (Lutensol ® A07) C10-Guerbet (2- 0 1.5 00 1.2 0 propylheptan-I-ol) alcohol ethoxylate with average 7 moles ofethoxylation (Lutensol ® XP70) C16-18 alcohol 0 0.5 0 0 0.3 0 ethoxylatewith average 7 moles of ethoxylation C12-18 alcohol 0 0.3 0 0 0 0ethoxylate with average 5 moles of ethoxylation C12-14 alkyl 0 0 0.70.54 0.1 1

HDD Detergent Compositions 1 2 3 4 5 6 (wt (wt (wt (wt (wt (wtIngredient %) %) %) %) %) %) hydroxyethyl dimethyl ammonium chloride(Praepagen ® HY) Sodium 0 0 0.6 0 1 0 tripolyphosphate Zeolite A(builder) 2.7 3.4 0 0 0.5 1.6 Citric Acid 1.8 2 0 1.4 0 2 Sodium citrate0 1.9 0 0 0 0 Sodium bicarbonate 29 35 36.7 34 53 22 Sodiumsesquicarbonate 0 0 1.2 0 0 0 dihydrate Sodium carbonate 1.2 0 1.9 0 0 0Sodium polyacrylate 0 0 1 0 0 0 (MW 4000, Sokalan PA25 CL) Sodiumpolyacrylate 1.45 1.6 0 0.97 1 0 (MW 8000, Sokalan PA30 CL) Sodium 0 00.3 0 0 3 polyacrylate/maleate copolymer MW 70,000, 70:30 ratio,Sokalan ® CPS Polyethylene 0 0 0.8 1 1 0 glycol/vinyl acetate randomgraft copolymer Carboxymethyl 1 0.9 0 0 0 0 cellulose (Finnfix ® GDA)Carboxymethyl 0 0 0 0.3 1.1 0.92 cellulose (Finnfix ® V) Hydrophobically0 0 0.5 0 0 0 modified carboxymethyl cellulose (Finnfix ® SH-1) C.I.Fluorescent 0.1 0.13 0.1 0.03 0.05 0.18 Brightener 260 C.I. Fluorescent0 0.06 0.08 0 0 0 Brightener 351 (Tinopal ® CBS) Diethylenetriamine 0 00.2 0.1 0.2 0 pentaacetic acid Tetrasodium S,S- 0 0 0 0.3 0 0.3ethylenediamine disuccinate Diethylenetriamine 0 0.2 0 0 0 0 penta(methylene phosphonic acid), heptasodium salt 1-Hydroxyethane-1,1- 0.10.2 0.3 0 0.2 0.4 diphosphonic acid

HDD Detergent Compositions 1 2 3 4 5 6 (wt (wt (wt (wt (wt (wtIngredient %) %) %) %) %) %) 2-Phosphono- 0 0 0 0.4 0 0 butane 1,2,4-tricarboxylic acid (Bayhibit ® AM) MgS04 0 0 0 0.8 0 0.4 Sodium 9 12 7 68 9 percarbonate Propylene 7 10 10.8 0 0 0 glycol diacetate Triethylene0 0 0 5 7 3.9 glycol diacetate Oxaziridinium- 0.03 0 0.03 0.02 0.05 0.02based bleach booster Protease 1 4.3 3.3 6.3 5.7 3.3 0 Protease 2 0 0 0 00 2.2 Amyalse 2.2 1.51 1 2.2 1.9 3.3 Lipase 0 0 3.6 0 0 2.7Endoglucanase 0 0 5.3 3.3 0 0 1 Endoglucanase 2.1 1.3 0 0 0 2.4 2Mannanase 1.3 1.54 1.3 0 1.2 1.9 Perhydrolase 1 2 0 1.8 0 2.1 1.9Perhydrolase 2 0 4.1 0 2.3 0 0 Direct Violet 9 0 0 0.0003 0.0004 0 0Solvent Violet 0 0 0.002 0 0 0 13 Texcare ® 0.3 1.2 0 1 0.33 0.3 SRA300FDye lock 0.02 0.02 0 0 0 0 (Tinolux ® 0 0 0 0 0 0.0015 BMC) C.I. FoodRed 0 0 0.001 0 0 0.001 14 Suds 0.2 0.2 0 0 0.3 0 suppressor granuleMoisture 7 6.3 8.9 9.1 4.3 4.6 Perfume 0.2 0.3 0.4 0.3 0.2 0.3 Sodiumsulfate Balance Balance Balance Balance Balance Balance to 100% to 100%to 100% to 100% to 100% to 100%

Automatic Dishwashing (ADW) Detergent Compositions Formulation 1 2 3 4Level Level Level Level Ingredient % wt % wt % wt % wt Solid ADWdetergent composition STPP 35 0 0 56 Carbonate 24 45 40 18.5Methylglycine diacetic acid (83% active) 0 15 20 0 Silicate 7 7 7 1.5TEAD (Tetraacety lethy lenediamine) 0.5 0.5 0.5 3.8 Zinc carbonate 0.50.5 0.5 0 SLF18 1.5 1.5 1.5 0 Plurafac LF224 0.6 Penta AmineAcetato-cobalt(III) 0.5 0.5 0.5 0.6 nitrate (1% active) Percarbonate 1515 15 11 Sulphonated polymer 10 4 3 5.1 Amylase (14.4 mg/g active) 1.31.8 1.5 0.7 Processing aids, perfume and To To To To sodium sulphatebalance balance balance balance Liquid automatic dishwashing detergentcomposition Dipropylene glycol 45 45 45 25 SLF18 45 45 45 0 Neodol1-9 33 3 2.6 Lutensol T07 30 Plurafac LF224 32.4 Amine Oxide 3.6 Glycerine 22 2 4 Processing aids and Dyes To To To To balance balance balancebalance Second Liquid automatic dishwashing detergent composition (partof three compartment unit dose)

HDL Detergent Compositions Formulations Compound I II III IV V LAS 24 326 3 6 NaC₁₆-C₁₇ HSAS — — — 5 — C₁₂-C₁₅ AE_(1.8)S — — 8 7 5 C₈-C₁₀ propyl2 2 2 2 1 dimethyl amine C₁₂-C₁₄ alkyl — — — — 2 dimethyl amine oxideC₁₂-C₁₅ AS alkyl — — 17 — 8 sulphate C₁₂-C₁₄ alkyl N- — 5 4 4 3 methylglucamide (CFAA) surfactant C₁₂-C₁₄ Fatty 12 6 1 1 1 alcohol ethoxylateC₁₂-C₁₈ Fatty acid 3 — 4 2 3 Citric acid 4.5 5 3 2 1 (anhydrous) DETPMP— — 1 1 0.5 Monoethanolamine 5 5 5 5 2 Sodium hydroxide — — 2.5 1 1.5 1NHCl aqueous #1 #1 — — — solution Propanediol 12.7 14.5 13.1 10 8 Ethanol1.8 2.4 4.7 5.4 1 DTPA 0.5 0.4 0.3 0.4 0.5 Pectin Lyase — — — 0.005 —Amylase 0.001 0.002 — — — Cellulase — — 0.0002 — 0.0001 Lipase 0.1 — 0.1— 0.1 Metalloprotease 1 0.05 0.3 — 0.5 0.2 (optional) Metalloprotease 2— — 0.08 — — Protease A — — — — 0.1 (optional) Aldose Oxidase — — 0.3 —0.003 ZnCl2 0.1 0.05 0.05 0.05 0.02 Ca formate 0.05 0.07 0.05 0.06 0.07DETBCHD — — 0.02 0.01 — SRP1 (anionically 0.5 0.5 — 0.3 0.3 end cappedpolyesters) Boric acid — — — — 2.4 Sodium xylene — — 3 — — sulfonateSodium cumene — — — 0.3 0.5 sulfonate DC 3225C 1 1 1 1 1 2-butyl-octanol0.03 0.04 0.04 0.03 0.03 Brightener 1 0.12 0.1 0.18 0.08 0.1 Balance to100% perfume/dye and/or water #1: Add 1N HCl aq. soln to adjust the neatpH of the formula in the range from about 3 to about 5. The pH ofExamples above (I)-(II) is about 5 to about 7, and of (III)-(V) is about7.5 to about 8.5.

HDL Detergent Compositions Formulations Compound I II III IV V VI LAS11.5 11.5 9 — 4 — C₁₂-C₁₅AE_(2.85)S — — 3 18 — 16 C₁₄-C₁₅E_(2.5)S 11.511.5 3 — 16 — C₁₂-C₁₃E₉ — — 3 2 2 1 C₁₂-C₁₃E₇ 3.2 3.2 — — — — C12-C14alkyl N-methyl — — — 5 — 3 glucamide (CFAA) surfactant TPKFA (C12-C14topped 2 2 — 2 0.5 2 whole cut fatty acids) Citric Acid (Anhydrous) 3.23.2 0.5 1.2 2 1.2 Ca formate 0.1 0.1 0.06 0.1 — — Na formate 0.5 0.50.06 0.1 0.05 0.05 ZnCl2 0.1 0.05 0.06 0.03 0.05 0.05 Sodium CumeneSulfonate 4 4 1 3 1.2 — Borate 0.6 0.6 1.5 — — — Sodium Hydroxide 6 6 23.5 4 3 Ethanol 2 2 1 4 4 3 1,2 Propanediol 3 3 2 8 8 5 Monoethanolamine3 3 1.5 1 2.5 1 TEPAE (tetraethylene 2 2 — 1 1 1 pentaamine ethoxylate)Metalloprotease 1 (optional) 0.03 0.05 — 0.03 — 0.02 Metalloprotease 2 —— 0.01 — 0.08 — Protease A (optional) — — 0.01 — — —

HDL Detergent Compositions Formulations Compound I II III IV V VI Lipase— — —  0.002 — — Amylase — — — —  0.002 — Cellulase — — — — —  0.0001Pectin Lyase  0.005  0.005 — — — Aldose Oxidase 0.05 — —  0.05 — 0.02Galactose — 0.04 — oxidase pentaamine 0.03 0.03 0.02 — — — acetatecobalt (III) salt PAAC DETBCHD — — —  0.02 0.01 — SRP1 0.2  0.2  — 0.1 —— (anionically end capped polyesters) DTPA — — — 0.3 — — polyvinyl — — —0.3 — 0.2 pyridine-N- Oxide (PVNO) Brightener 1 0.2  0.2  0.07 0.1 — —Silicone 0.04 0.04 0.02 0.1 0.1  0.1 antifoam Balance to 100%perfume/dye and/or water

Liquid Hand Dishwashing (Hand Dish Liquid) Detergent CompositionsFormulations Compound I II III IV V VI C₁₂-C₁₅ AE_(1.8)S 30 28 25 — 1510 LAS — — — 5 15 12 Paraffin Sulfonate — — — 20 — — C₁₀-C₁₈ Alkyl 5 3 7— — — Dimethyl Amine Oxide Betaine 3 — 1 3 1 — C₁₂ poly-hydroxy — — — 3— 1 fatty acid amide C₁₄ poly-OH — 1.5 — — — — fatty acid amide C₁₁E₉ 2— 4 — — 20 DTPA — — — — 0.2 — Tri-sodium Citrate 0.25 — — 0.7 — —dihydrate (builder) Diamine (Dimethyl 1 5 7 1 5 7 aminopropyl amine;1,6-hezane diamine; 1,3-propane diamine; 2-methyl-1,5- pentane diamine;1,3- pentanediamine; 1-methyl- diaminopropane) MgCl₂ 0.25 — — 1 — —Metalloprotease 1 0.02 0.01 — 0.01 — 0.05 (optional) Metalloprotease 2 —— 0.03 — 0.02 — Protease A (optional) — 0.01 — — — — Amylase 0.001 — —0.002 — 0.001 Aldose Oxidase 0.03 — 0.02 — 0.05 — Sodim Cumene — — — 21.5 3 Sulfonate pentaamine acetate 0.01 0.01 0.02 — — — cobalt (III)salt DETBCHD — — — 0.01 0.02 0.01 Balance to 100% perfume/dye and/orwater The pH of Examples (I)-(VI) is about 8 to about 11.

Liquid Automatic Dish Washing Detergent Compositions FormulationsCompound I II III IV V STPP (sodium 16.00 16.00 18.00 16.00 16.00tripoly phosphate) Potassium Sulfate — 10.00 8.00 — 10.00 1,2propanediol 6.00 0.50 2.00 6.00 0.50 Boric Acid — — — 4.00 3.00 CaCl₂dihydrate 0.04 0.04 0.04 0.04 0.04 Nonionic surfactant 0.50 0.50 0.500.50 0.50 Metalloprotease 1 0.10 0.03 — 0.03 — (optional)Metalloprotease 2 — — 0.05 — 0.06 Protease B — — — 0.01 — (optional)Amylase 0.02 — 0.02 0.02 — Aldose Oxidase — 0.15 0.02 — 0.01 GalactoseOxidase — — 0.01 — 0.01 pentaamine acetate 0.01 — — 0.01 — cobalt (III)salt PAAC (bleach catalyst) DETBCHD — 0.01 — — 0.01 Balance to 100%perfume/dye and/or water

Granular and/or Tablet Detergent Compositions Formulations Compound I IIIII IV V C₁₄-C₁₅AS or TAS 8 5 3 3 3 (sodium tallow alkyl sulfate) LAS 8— 8 — 7 C₁₂-C₁₅AE₃S 0.5 2 1 — — C₁₂-C₁₅E₅ or E₃ 2 — 5 2 2 QAS(quarternary — — — 1 1 ammonium salt) Zeolite A 20 18 11 — 10 SKS-6 (dryadd) — — 9 — — (layered silicate) MA/AA (acrylate/maleate 2 2 2 — —copolymer) AA (polyacrylate — — — — 4 polymer) 3Na Citrate 2H₂O — 2 — —— Citric Acid (Anhydrous) 2 — 1.5 2 — DTPA 0.2 0.2 — — — EDDS — — 0.50.1 — HEDP — — 0.2 0.1 — PB1 (sodium perborate 3 4.8 — — 4 monohydrate)Percarbonate — — 3.8 5.2 — NOBS 1.9 — — — — NACA OBS — — 2 — — TAED 0.52 2 5 1 BB1 (3-(3,4- 0.06 — 0.34 — 0.14 Dihydroisoquinolinium) propanesulfonate (DIPS)) BB2 3-(3,4- — 0.14 — 0.2 — Dihydroisoquinolinium)-decane-2-sulfate Anhydrous sodium 15 18 — 15 15 carbonate Sulfate 5 12 517 3 Silicate — 1 — — 8 Metalloprotease 1 0.03 — 0.1 0.06 — (optional)Metalloprotease 2 — 0.05 — — 0.1 Protease B (optional) — 0.01 — — —Protease C (optional) — — — 0.01 — Lipase — 0.008 — — — Amylase 0.001 —— — 0.001 Cellulase — 0.0014 — — — Pectin Lyase 0.001 0.001 0.001 0.0010.001 Aldose Oxidase 0.03 — 0.05 — — pentaamine acetate — 0.01 — — 0.05cobalt (III) salt PAAC Balance to 100% Moisture and/or Minors* *Perfume,dye, brightener/SRP1/Nacarboxymethylcellulose/photobleach/MgSO4/PVPVI/suds suppressor/highmolecular PEG/clay.

High Density Automatic Dish Washing Detergent Compositions FormulationsCompound I II III IV V VI STPP (sodium tripoly — 45 45 — — 40 phosphate)3Na Citrate 2H₂O 17 — — 50 40.2 — Na Carbonate 17.5 14 20 — 8 33.6Bicarbonate — — — 26 — — Silicate 15 15 8 — 25 3.6 Metasilicate 2.5 4.54.5 — — — PB1 (sodium perborate — — 4.5 — — — monohydrate) PB4 (sodiumperborate — — — 5 — — tetrahydrate) Percarbonate — — — — — 4.8 BB1(3-(3,4- — 0.1 0.1 — 0.5 — Dihydroisoquinolinium)propane sulfonate(DIPS)) BB2 3-(3,4- 0.2 0.05 — 0.1 — 0.6 Dihydroisoquinolinium)-decane-2-sulfate Nonionic detergent 2 1.5 1.5 3 1.9 5.9 HEDP 1 — — — — —DETPMP 0.6 — — — — — pentaamine acetate cobalt (III) 0.03 0.05 0.02 — —— salt PAAC Paraffin oil Winog 70 0.5 0.4 0.4 0.6 — — Metalloprotease 1(optional) 0.072 0.053 — 0.026 — 0.01 Metalloprotease 2 — — 0.053 —0.059 — Protease B (optional) — — — — — 0.01 Amylase 0.012 — 0.012 —0.021 0.006 Lipase — 0.001 — 0.005 — — Pectin Lyase 0.001 0.001 0.001 —— — Aldose Oxidase 0.05 0.05 0.03 0.01 0.02 0.01 BTA (benzotriazole) 0.30.2 0.2 0.3 0.3 0.3 Polycarboxylate 6 — — — 4 0.9 Perfume 0.2 0.1 0.10.2 0.2 0.2 Balance to 100% Moisture and/or Minors**Brightener/dye/SRP1/Nacarboxymethylcellulose/photobleach/MgSO4/PVPVI/suds suppressor/highmolecular PEG/clay. The pH of Examples (I) through (VI) is from about9.6 to about 11.3.

Tablet Detergent Compositions Formulations Compound I II III IV V VI VIIVIII STPP (sodium tripoly — 48.8 44.7 38.2 — 42.4 46.1 46 phosphate) 3NaCitrate 2H₂O 20 — — — 35.9 — — — Na Carbonate 20 5 14 15.4 8 23 20 —Silicate 15 14.8 15 12.6 23.4 2.9 4.3 4.2 Lipase 0.001 — 0.01 — 0.02 — —— Protease B 0.01 — — — — — — — Protease C — — — — — 0.01 — —Metalloprotease 1 (optional) 0.01 0.08 — 0.04 — 0.023 — 0.05Metalloprotease 2 — — 0.05 — 0.052 — 0.023 — Amylase 0.012 0.012 0.012 —0.015 — 0.017 0.002 Pectin Lyase 0.005 — — 0.002 — — — — Aldose Oxidase— 0.03 — 0.02 0.02 — 0.03 — PB1 (sodium perborate — — 3.8 — 7.8 — — 4.5monohydrate) Percarbonate 6 — — 6 — 5 — — BB1 (3-(3,4- 0.2 — 0.5 — 0.30.2 — — Dihydroisoquinolinium)propane sulfonate (DIPS)) BB2 3-(3,4- —0.2 — 0.5 — — 0.1 0.2 Dihydroisoquinolinium)- decane-2-sulfate Nonionicsurfactant 1.5 2 2 2.2 1 4.2 4 6.5 pentaamine acetate cobalt (III) 0.010.01 0.02 — — — — — salt PAAC DETBCHD — — — 0.02 0.02 — — — TAED — — — —— 2.1 — 1.6 HEDP 1 — — 0.9 — 0.4 0.2 — DETPMP 0.7 — — — — — — — Paraffinoil Winog 70 0.4 0.5 0.5 0.5 — — 0.5 — BTA (benzotriazole) 0.2 0.3 0.30.3 0.3 0.3 0.3 — Polycarboxylate 4 — — — 4.9 0.6 0.8 — PEG 400-30,000 —— — — — 2 — 2 Glycerol — — — — — 0.4 — 0.5 Perfume — — — 0.05 0.2 0.20.2 0.2 Balance to 100% Moisture and/or Minors* *Brightener/SRP1/Nacarboxymethylcellulose/photobleach/MgSO4/PVPVI/suds suppressor/highmolecular PEG/clay. The pH of Examples (I) through (VII) is from about10 to about 11.5; pH of (VIII) is from 8-10. The tablet weight ofExamples (I) through (VIII) is from about 20 grams to about 30 grams.

Liquid Hard Surface Detergent Compositions Formulations Compound I IIIII IV V VI VII C₉-C₁₁E₅ 2.4 1.9 2.5 2.5 2.5 2.4 2.5 C₁₂-C₁₄E₅ 3.6 2.92.5 2.5 2.5 3.6 2.5 C₇-C₉E₆ — — — — 8 — — C₁₂-C₁₄E₂₁ 1 0.8 4 2 2 1 2 LAS— — — 0.8 0.8 — 0.8 Sodim Cumene 1.5 2.6 — 1.5 1.5 1.5 1.5 SulfonateIsachem ® AS 0.6 0.6 — — — 0.6 — (branched alcohol alkyl sulfate) Na₂CO₃0.6 0.13 0.6 0.1 0.2 0.6 0.2 3Na Citrate 2H₂O 0.5 0.56 0.5 0.6 0.75 0.50.75 NaOH 0.3 0.33 0.3 0.3 0.5 0.3 0.5 Fatty Acid 0.6 0.13 0.6 0.1 0.40.6 0.4 2-butyl octanol 0.3 0.3 — 0.3 0.3 0.3 0.3 PEG DME-2000 ® 0.4 —0.3 0.35 0.5 — — PVP (vinylpyrrolidone 0.3 0.4 0.6 0.3 0.5 — —homopolymer) MME PEG (2000) ® — — — — — 0.5 0.5 Jeffamine ® ED-2001 —0.4 — — 0.5 — — (capped polyethylene glycol) pentaamine acetate — — —0.03 0.03 0.03 — cobalt (III) salt PAAC DETBCHD 0.03 0.05 0.05 — — — —Metalloprotease 1 0.07 — 0.08 0.03 — 0.01 0.04 (optional)Metalloprotease 2 — 0.05 — — 0.06 — — Protease B (optional) — — — — —0.01 — Amylase 0.12 0.01 0.01 — 0.02 — 0.01 Lipase — 0.001 — 0.005 —0.005 — Pectin Lyase 0.001 — 0.001 — — — 0.002 ZnCl2 0.02 0.01 0.03 0.050.1 0.05 0.02 Calcium Formate 0.03 0.03 0.01 — — — — PB1 (sodiumperborate — 4.6 — 3.8 — — — monohydrate) Aldose Oxidase 0.05 — 0.03 —0.02 0.02 0.05 Balance to 100% perfume/dye and/or water The pH ofExamples (I) through (VII) is from about 7.4 to about 9.5.

HDL Detergent Compositions Composition (wt % of composition) Ingredient1 2 3 4 C₁₂₋₁₅ 14.7 11.6 16.31 Alkylethoxy(1.8)sulfate C_(11.8)Alkylbenzene sulfonate 4.3 11.6 8.3 7.73 C₁₆₋₁₇ Branched alkyl sulfate1.7 1.29 3.09 C₁₂₋₁₄ Alkyl-9-ethoxylate 0.9 1.07 1.31 C₁₂ dimethylamineoxide 0.6 0.64 1.03 Citric acid 3.5 0.65 3 0.66 C₁₂₋₁₈ fatty acid 1.52.32 3.6 1.52 Sodium Borate (Borax) 2.5 2.46 1.2 2.53 Sodium C₁₂₋₁₄alkyl ethoxy 3 2.9 sulfate C₁₄₋₁₅ alkyl 7-ethoxylate 4.2 C₁₂₋₁₄Alkyl-7-ethoxylate 1.7 Ca formate 0.09 0.09 0.09 A compound having the1.2 following general structure: bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)— bis((C₂H₅O)(C₂H₄O)n), wherein n = from 20 to30, and x = from 3 to 8, or sulphated or sulphonated variants thereofRandom graft co-polymer¹ 1.46 0.5 Ethoxylated 1.5 1.29 1.44Polyethylenimine ² Diethylene triamine 0.34 0.64 0.34 pentaacetic acidDiethylene triamine 0.3 penta(methylene phosphonic acid) Tinopal AMS-GX0.06 Tinopal CBS-X 0.2 0.17 0.29 Amphiphilic alkoxylated 1.28 1 0.4 1.93grease cleaning polymer ³ Ethanol 2 1.58 1.6 5.4 Propylene Glycol 3.93.59 1.3 4.3 Diethylene glycol 1.05 1.54 1.15 Polyethylene glycol 0.060.04 0.1 Monoethanolamine 3.05 2.41 0.4 1.26 NaOH 2.44 1.8 3.01 SodiumCumene Sulphonate 1 Sodium Formate 0.11 0.09 Water, Aesthetics (Dyes,perfumes) and Minors (Enzymes, solvents, structurants) balance balancebalance balance ¹Random graft copolymer is a polyvinyl acetate graftedpolyethylene oxide copolymer having a polyethylene oxide backbone andmultiple polyvinyl acetate side chains. The molecular weight of thepolyethylene oxide backbone is about 6000 and the weight ratio of thepolyethylene oxide to polyvinyl acetate is about 40 to 60 and no morethan 1 grafting point per 50 ethylene oxide units. ² Polyethylenimine(MW = 600) with 20 ethoxylate groups per —NH. ³ Amphiphilic alkoxylatedgrease cleaning polymer is a polyethylenimine (MW = 600) with 24ethoxylate groups per —NH and 16 propoxylate groups per —NH.

Light-Duty Liquid Dishwashing Detergent Compositions Composition 1 2 3 4Linear Alkylbenzene — — — Sulfonate (1) Alkyl Ethoxy Sulfate (2) 18%  17% 17%  18%  Paraffin Sulfonate (C15) — — — — CAP = coco amido — — 9%5% propyl Betaine Nonionic (3) — — 1% — Amine Oxide (4)  6% 5.50% — 4%Alkylpolyglucoside 4% Alcohol (5) — — 5% 7% Pura =  1% 0.80% — —polypropyleneglycol Citrate — — 0.30%   0.60%   Salt (6) 1.20%   1.00% —0.50%   SCS = sodium cumene — — 0.80%   — sulfonate glycerol 15%   5% 3%— Na-lactate — — — 5% cationic polymer (7) 0.10%   0.10% 0.30%   0.20%  Present amylase 0.0075 0.005 0.0025 0.03 Glycol distearate from 0.4 00.4 0 Euperlan ® Cognis Hydrogenated Castor Oil 0 0.1 0 0.1 Thixcin ®Elementis Mica (BASF Mearlin 0 0.05 0 0.05 superfine) Minors* Balance to100% with water pH 9 9 6 6 Optional Minors*: dyes, opacifier, perfumes,preservatives, hydrotropes, processing aids, and/or stabilizers. (1)Linear Alkylbenzene Sulfonate: LAS: C11.4 (2) Alkyl Ethoxy Sulfate:AExS: (3) Nonionic: AlkylEthoxylate (4) Di-methyl coco alkyl amine oxide(5) Alcohol: Ethanol (6) Salt: NaCl (7) cationically modifiedhydroxyethyl cellulose (Polyquaternium-10-UCARE LR-400 ex Amerchol).

Liquid laundry detergent compositions suitable for front-loadingautomatic washing machines Composition (wt % of composition) Ingredient1 2 3 4 5 6 7 8 Alkylbenzene sulfonic acid 7 11 4.5 1.2 1.5 12.5 5.2 4Sodium C₁₂₋₁₄ alkyl ethoxy 3 sulfate 2.3 3.5 4.5 4.5 7 18 1.8 2 C₁₄₋₁₅alkyl 8-ethoxylate 5 8 2.5 2.6 4.5 4 3.7 2 C₁₂ alkyl dimethyl amineoxide — — 0.2 — — — — — C₁₂₋₁₄ alkyl hydroxyethyl dimethyl — — — 0.5 — —— — ammonium chloride C₁₂₋₁₈ Fatty acid 2.6 4 4 2.6 2.8 11 2.6 1.5Citric acid 2.6 3 1.5 2 2.5 3.5 2.6 2 Protease* 0.05 0.03 0.04 0.03 0.040.03 0.03 0.02 Amylase 0.1 0.2 0.15 — 0.05 0.5 0.1 0.2 Mannanase 0.050.1 0.05 — — 0.1 0.04 — Random graft co-polymer¹ 1 0.2 1 0.4 0.5 2.7 0.31 A compound having the following general 0.4 2 0.4 0.6 1.5 1.8 0.7 0.3structure: bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)-bis((C₂H₅O)(C₂H₄O)n), wherein n = from 20 to 30, and x = from 3 to 8, orsulphated or sulphonated variants thereof Ethoxylated Polyethylenimine²— — — — — 0.5 — — Amphiphilic alkoxylated grease cleaning 0.1 0.2 0.10.2 0.3 0.3 0.2 0.3 polymer³ Diethoxylated poly (1,2 propylene — — — — —— 0.3 — terephthalate) Diethylenetriaminepenta(methylenephosphonic) 0.20.3 — — 0.2 — 0.2 0.3 acid Hydroxyethane diphosphonic acid — — 0.45 — —1.5 — 0.1 FWA (fluorescent whitening agent) 0.1 0.2 0.1 — — 0.2 0.05 0.1Solvents (1,2 propanediol, ethanol), 3 4 1.5 1.5 2 4.3 2 1.5Hydrogenated castor oil derivative 0.4 0.4 0.3 0.1 0.3 — 0.4 0.5 Boricacid 1.5 2.5 1.5 1.5 0.5 1.5 1.5 Na formate — — — 1 — — — — Reversibleprotease inhibitor⁴ — — 0.002 — — — — — Perfume 0.5 0.7 0.5 0.5 0.8 1.50.5 0.8 Perfume MicroCapsules slurry (30% am) 0.2 0.3 0.7 0.2 0.05 0.40.9 0.7 Ethoxylated thiophene Hueing Dye⁵ 0.005 0.007 0.01 0.008 0.0080.007 0.007 0.008 Buffers (sodium hydroxide, To pH 8.2 Monoethanolamine)Water and minors (antifoam, aesthetics) To 100% ¹Random graft copolymeris a polyvinyl acetate grafted polyethylene oxide copolymer having apolyethylene oxide backbone and multiple polyvinyl acetate side chains.The molecular weight of the polyethylene oxide backbone is about 6000and the weight ratio of the polyethylene oxide to polyvinyl acetate isabout 40 to 60 and no more than 1 grafting point per 50 ethylene oxideunits. ²Polyethylenimine (MW = 600) with 20 ethoxylate groups per —NH.³Amphiphilic alkoxylated grease cleaning polymer is a polyethylenimine(MW = 600) with 24 ethoxylate groups per —NH and 16 propoxylate groupsper —NH ⁵Ethoxylated thiophene Hueing Dye is as described in U.S. Pat.No. 7,208,459 B2. *Remark: all enzyme levels expressed as % enzyme rawmaterial, except for protease which is expressed as % of active proteinadded to the product.. ⁴Reversible Protease inhibitor of structure shownbelow the Table.

Liquid laundry detergent compositions suitable for top-loading automaticwashing machines Composition (wt % of composition) Ingredient 1 2 3 4 56 7 8 C₁₂₋₁₅ 20.1 15.1 20 15.1 13.7 16.7 10 9.9 Alkylethoxy(1.8)sulfateC_(11.8) Alkylbenzene 2.7 2 1 2 5.5 5.6 3 3.9 sulfonate C₁₆₋₁₇ Branchedalkyl 6.5 4.9 4.9 3 9 2 sulfate C₁₂₋₁₄ Alkyl-9-ethoxylate 0.8 0.8 0.80.8 8 1.5 0.3 11.5 C₁₂ dimethylamine oxide 0.9 Citric acid 3.8 3.8 3.83.8 3.5 3.5 2 2.1 C₁₂₋₁₈ fatty acid 2 1.5 2 1.5 4.5 2.3 0.9 Protease*0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 Amylase 1 0.7 0.3 0.6 0.3 0.6 0.4Amylase 2 1.1 Mannanase 0.1 0.1 Pectate Lyase 0.1 0.2 Borax 3 3 2 3 33.3 Na & Ca formate 0.2 0.2 0.2 0.2 0.7 A compound having the 1.6 1.6 31.6 2 1.6 1.3 1.2 following general structure:bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)- bis((C₂H₅O)(C₂H₄O)n),wherein n = from 20 to 30, and x = from 3 to 8, or sulphated orsulphonated variants thereof Random graft co-polymer¹ 0.4 0.2 1 0.5 0.61 0.8 1 Diethylene triamine 0.4 0.4 0.4 0.4 0.2 0.3 0.8 pentaacetic acidTinopal AMS-GX 0.2 0.2 0.2 0.2 0.2 0.3 0.1 (brightener) Tinopal CBS-X0.1 0.2 (brightener) Amphiphilic alkoxylated 1 1.3 1.3 1.4 1 1.1 1 1grease cleaning polymer³ Texcare 240N (Clariant) 1 Ethanol 2.6 2.6 2.62.6 1.8 3 1.3 Propylene Glycol 4.6 4.6 4.6 4.6 3 4 2.5 Diethylene glycol3 3 3 3 3 2.7 3.6 Polyethylene glycol 0.2 0.2 0.2 0.2 0.1 0.3 0.1 1.4Monoethanolamine 2.7 2.7 2.7 2.7 4.7 3.3 1.7 0.4 Triethanolamine 0.9NaOH to pH to pH to pH to pH to pH to pH to pH to pH 8.5 8.3 8.3 8.3 8.38.3 8.3 8.3 Suds suppressor Dye 0.01 0.01 0.01 0.01 0.01 0.01 0 Perfume0.5 0.5 0.5 0.5 0.7 0.7 0.8 0.6 Perfume MicroCapsules 0.2 0.5 0.2 0.30.1 0.3 0.9 1 slurry (30% am) Ethoxylated thiophene 0.003 0.002 0.0020.005 0.002 0.004 0.004 0.003 Hueing Dye⁵ Water balance balance balancebalance balance balance balance balance ¹Random graft copolymer is apolyvinyl acetate grafted polyethylene oxide copolymer having apolyethylene oxide backbone and multiple polyvinyl acetate side chains.The molecular weight of the polyethylene oxide backbone is about 6000and the weight ratio of the polyethylene oxide to polyvinyl acetate isabout 40 to 60 and no more than 1 grafting point per 50 ethylene oxideunits. ³Amphiphilic alkoxylated grease cleaning polymer is apolyethylenimine (MW = 600) with 24 ethoxylate groups per —NH and 16propoxylate groups per —NH ⁵Ethoxylated thiophene Hueing Dye is asdescribed in U.S. Pat. No. 7,208,459 B2. *Remark: all enzyme levelsexpressed as % enzyme raw material, except for protease which isexpressed as % of active protein added to the product..

Granular detergent compositions Component 1 2 3 4 5 6 Linearalkylbenzenesulfonate with 15 12 20 10 12 13 aliphatic carbon chainlength C₁₁-C₁₂ Other surfactants 1.6 1.2 1.9 3.2 0.5 1.2 Phosphatebuilder(s) 2 3 4 Zeolite 1 1 4 1 Silicate 4 5 2 3 3 5 Sodium Carbonate 25 5 4 0 3 Polyacrylate (MW 4500) 1 0.6 1 1 1.5 1 Carboxymethyl cellulose(Finnfix 1 — 0.3 — 1.1 — BDA ex CPKelco) Cellulase 0.23 0.17 0.5 0.2 0.20.6 Protease 0.23 0.17 0.5 0.2 0.2 0.6 Amylase 0.23 0.17 0.5 0.2 0.2 0.6Fluorescent Brightener(s) 0.16 0.06 0.16 0.18 0.16 0.16Diethylenetriamine pentaacetic acid or 0.6 0.6 0.25 0.6 0.6 Ethylenediamine tetraacetic acid MgSO₄ 1 1 1 0.5 1 1 Bleach(es) and Bleachactivator(s) 6.88 6.12 2.09 1.17 4.66 Ethoxylated thiophene Hueing Dye⁵0.002 0.001 0.003 0.003 — — Direct Violet 9 ex Ciba Specialty 0.00060.0004 0.0006 Chemicals Sulfate/Citric Acid/Sodium Balance to 100%Bicarbonate/Moisture/perfume ⁵Ethoxylated thiophene Hueing Dye is asdescribed in U.S. Pat. No. 7,208,459 B2.

Granular Laundry Detergent Compositions and Their Components DetergentCompositions Component 1 2 3 4 5 6 Linear alkylbenzenesulfonate with 1512 20 10 12 13 aliphatic carbon chain length C₁₁-C₁₂ Other surfactants1.6 1.2 1.9 3.2 0.5 1.2 Phosphate builder(s) 2 3 4 Zeolite 1 1 4 1Silicate 4 5 2 3 3 5 Sodium Carbonate 2 5 5 4 0 3 Polyacrylate (MW 4500)1 0.6 1 1 1.5 1 Carboxymethyl cellulose 1 — 0.3 — 1.1 — Cellulase (15.6mg/g) 0.23 0.17 0.5 0.2 0.2 0.6 Protease 0.23 0.17 0.05 0.2 0.03 0.1Amylase (14 mg/g) 0.23 0.17 0.5 0.2 0.2 0.6 Mannanase (4 mg/g) 0.1 0.10.1 Lipase (18.6 mg/g) 0.2 0.1 0.3 Fluorescent Brightener(s) 0.16 0.060.16 0.18 0.16 0.16 Diethylenetriamine pentaacetic acid or 0.6 0.6 0.250.6 0.6 Ethylene diamine tetraacetic acid MgSO₄ 1 1 1 0.5 1 1 Bleach(es)and Bleach activator(s) 6.88 6.12 2.09 1.17 4.66 Ethoxylated thiopheneHueing Dye⁵ 0.002 0.001 0.003 0.003 — — Direct Violet 9 ex CibaSpecialty 0.0006 0.0004 0.0006 Chemicals Sulfate/Citric Acid/SodiumBicarbonate/ Balance to 100% Moisture/perfume ⁵Ethoxylated thiopheneHueing Dye is as described in U.S. Pat. No. 7,208,459 B2.

Granular Laundry Detergent Compositions and Their Components DetergentComposition Component 7 8 9 10 11 Surfactants C₁₆₋₁₇ Branched alkylsulfate 3.55 15.8 C₁₂₋₁₄ alkyl sulphate 1.5 Sodium linear 9.6 10.6 7.5 9alkylbenzenesulfonate with aliphatic chain length C₁₁-C₁₂ SodiumC_(14/15) alcohol ethoxy- 1.15 2.88 3-sulfate Sodium C_(14/15) alkylsulphate 2.37 C_(14/15) alcohol ethoxylate with 1.17 1 average 7 molesof ethoxylation mono-C₈₋₁₀ alkyl mono- 0.45 hydroxyethyl di-methylquaternary ammonium chloride Di methyl hydroxyl ethyl lauryl 0.18ammonium chloride Zeolite A 13.9 4.7 0.01 2.9 1.8 Sodium Silicate1.6.ratio 4 0.2 4 4 Sodium Silicate 2.35.ratio 8 Citric Acid 2.5 1.4Sodium tripolyphosphate 5 Sodium Carbonate 24.1 30 16.9 24.4 21Nonanoyloxybenzenesuplhonate 5.78 2.81 0.96 Oxaziridinium-based bleach0.03 0.017 booster Tetrasodium S,S,- 0.2 ethylenediaminedisuccinateDiethylenetriamine penta 0.61 0.33 (methylene phosphonic acid),heptasodium salt Hydroxyethane dimethylene 0.29 0.45 phosphonic acidEthylene diamine tetraacetate 0.27 MgSO4 0.47 0.5994 0.782 SodiumPercarbonate 7 4.4 15.9 19.1 Tetra Acetyl Ethylene Diamine 3.3 4.6Sodium Perborate Monohydrate 1.2 Carboxymethyl cellulose 0.1 0.17 1.690.23 (e.g., Finnfix BDA ex CPKelco) Sodium Acrylic acid/maleic 0.02363.8 2 2.5 acid co-polymer (70/30) Sodium polyacrylate (Sokalan 4 0.84PA30 CL) Terephthalate polymer 0.23 Polyethylene glycol/vinyl 0.89 0.890.91 acetate random graft co polymer Photobleach-zinc 0.005 0.001 0.002phthalocyanine tetrasulfonate C.I. Fluorescent Brightener 260 0.11 0.150.04 0.23 0.15 C.I. Fluorescent Brightener 351 0.1 (Tinopal ® CBS)

Granular Laundry Detergent Compositions and Their Components DetergentComposition Component 7 8 9 10 11 Suds suppressor 0.25 0.07 0.04 granuleHydrophobically 0.019 0.028 modified carboxy methyl cellulose(Finnifix ® SH-1) Bentonite 8.35 Miscellaneous (Dyes, Balance BalanceBalance Balance Balance perfumes, process aids, moisture and sodiumsulphate)

Unit Dose Detergent Compositions Ingredients 1 2 3 4 5 Alkylbenzene 14.514.5 14.5 14.5 14.5 sulfonic acid C 11- 13, 23.5% 2-phenyl isomer C₁₂₋₁₄alkyl ethoxy 3 7.5 7.5 7.5 7.5 7.5 sulfate C₁₂₋₁₄ alkyl 7- 13 13 13 1313 ethoxylate Citric Acid 0.6 0.6 0.6 0.6 0.6 Fatty Acid 14.8 14.8 14.814.8 14.8 Enzymes (as % raw 1.7 1.7 1.7 1.7 1.7 material not active)Present amylase (as 0.05 0.1 0.02 0.03 0.03 % active) Ethoxylated 4 4 44 4 Polyethylenimine¹ Series 1 GG36 0.02 0 0.01 0.02 0.03 protease (as %active) Hydroxyethane 1.2 1.2 1.2 1.2 1.2 diphosphonic acid Brightener0.3 0.3 0.3 0.3 0.3 P-diol 15.8 13.8 13.8 13.8 13.8 Glycerol 6.1 6.1 6.16.1 6.1 MEA 8 8 8 8 8 (monoethanolamide) brightener stabilizer TIPA — —2 — — (triisopropanolamine) TEA — 2 — — — (triethanolamine) Cumenesulphonate — — — — 2 cyclohexyl — — — 2 — dimethanol Water 10 10 10 1010 Structurant 0.14 0.14 0.14 0.14 0.14 Perfume 1.9 1.9 1.9 1.9 1.9Buffers To pH 8.0 (monoethanolamine) Solvents (1,2 To 100% propanediol,ethanol) ¹Polyethylenimine (MW = 600) with 20 ethoxylate groups per —NH.

Multiple Compartment Unit Dose Detergent Compositions Base Composition 1Ingredients % Glycerol (min 99) 5.3 1,2-propanediol 10 Citric Acid 0.5Monoethanolamine 10 Caustic soda — Dequest 2010 1.1 Potassium sulfite0.2 Nonionic Marlipal C24EO7 20.1 HLAS (surfactant) 24.6 Opticalbrightener FWA49 0.2 C12-15 Fatty acid 16.4 Polymer Lutensit Z96 2.9Polyethyleneimine ethoxylate 1.1 PEI600 E20 MgCl2 0.2 Solvents (1,2propanediol, ethanol) To 100%

Multi-compartment formulations Composition 1 2 Compartment A B C A B CVolume of each 40 ml 5 ml 5 ml 40 ml 5 ml 5 ml compartment Activematerial in Wt. % Perfume 1.6 1.6 1.6 1.6 1.6 1.6 Dyes <0.01 <0.01 <0.01<0.01 <0.01 <0.01 TiO2 0.1 — — — 0.1 — Sodium Sulfite 0.4 0.4 0.4 0.30.3 0.3 Acusol 305, 1.2 2 — — Rohm&Haas Hydrogenated castor 0.14 0.140.14 0.14 0.14 0.14 oil Base Composition 1 Add Add to Add Add to Add toAdd to to 100% to 100% 100% 100% 100% 100%

Phosphate-Free Detergent: IEC-60436 WFK Type B (pH = 10.4 in 3 g/l)Component Wt % Sodium citrate dehydrate 30 Maleic acid/Acrylic acid 12copolymer sodium Salt SOKALAN ® CP5 BASF Sodium perborate 5 monohydrateTAED 2 Sodium disilicate: Protil A 25 (Cognis) Linear fatty alcohol 2ethoxylate Sodium carbonate add to 100 anhydrous

Phosphate-Containing Detergent: IEC-60436 WFK Type C (pH = 10.5 in 3g/l) Component Wt % Sodium tripolyphosphate 23 Sodium citrate dehydrate22.3 Maleic acid/Acrylic acid 4 copolymer sodium salt Sodium perborate 6monohydrate TAED 2 Sodium disilicate: Protil A 5 (Cognis) Linear fattyalcohol 2 ethoxylate Sodium carbonate add to 100 anhydrous

Liquid laundry detergent compositions suitable for top-loading automaticwashing machines (1 &2) and front loading washing machines (3).Composition (wt % of composition) Ingredient 1 2 3 C₁₂₋₁₅Alkylethoxy(1.8)sulfate 14.7 11.6 C_(11.8) Alkylbenzene sulfonate 4.311.6 8.3 C₁₆₋₁₇ Branched alkyl sulfate 1.7 1.29 C₁₂₋₁₄Alkyl-9-ethoxylate 0.9 1.07 C₁₂ dimethylamine oxide 0.6 0.64 Citric acid3.5 0.65 3 C₁₂₋₁₈ fatty acid 1.5 2.32 3.6 Sodium Borate (Borax) 2.5 2.461.2 Sodium C₁₂₋₁₄ alkyl ethoxy 3 sulfate 2.9 C₁₄₋₁₅ alkyl 7-ethoxylate4.2 C₁₂₋₁₄ Alkyl-7-ethoxylate 1.7 Ca formate 0.09 0.09 A compound havingthe following general structure: 1.2bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)— bis((C₂H₅O)(C₂H₄O)n),wherein n = from 20 to 30, and x = from 3 to 8, or sulphated orsulphonated variants thereof Random graft co-polymer¹ 1.46 0.5Ethoxylated Polyethylenimine ² 1.5 1.29 Diethylene triamine pentaaceticacid 0.34 0.64 Diethylene triamine penta(methylene phosphonic acid) 0.3Tinopal AMS-GX 0.06 Tinopal CBS-X 0.2 0.17 Amphiphilic alkoxylatedgrease cleaning polymer ³ 1.28 1 0.4 Ethanol 2 1.58 1.6 Propylene Glycol3.9 3.59 1.3 Diethylene glycol 1.05 1.54 Polyethylene glycol 0.06 0.04Monoethanolamine 3.05 2.41 0.4 NaOH 2.44 1.8 Sodium Cumene Sulphonate 1Sodium Formate 0.11 Water, Aesthetics (Dyes, perfumes) and Minors(Enzymes, solvents, balance balance balance structurants) ¹Random graftcopolymer is a polyvinyl acetate grafted polyethylene oxide copolymerhaving a polyethylene oxide backbone and multiple polyvinyl acetate sidechains. The molecular weight of the polyethylene oxide backbone is about6000 and the weight ratio of the polyethylene oxide to polyvinyl acetateis about 40 to 60 and no more than 1 grafting point per 50 ethyleneoxide units. ² Polyethylenimine (MW = 600) with 20 ethoxylate groups per—NH. ³ Amphiphilic alkoxylated grease cleaning polymer is apolyethylenimine (MW = 600) with 24 ethoxylate groups per —NH and 16propoxylate groups per —NH

Granular laundry detergent compositions suitable for top-loadingautomatic washing machines (1-3) and front loading washing machines(4-5). The present amylase is separately added to these formulations.Ingredients 1 2 3 4 5 C₁₆₋₁₇ Branched alkyl sulfate 3.55 C₁₂₋₁₄ alkylsulphate 1.5 Sodium linear alkylbenzenesulfonate 9.6 15.8 10.6 7.5 9with aliphatic chain length C₁₁-C₁₂ Sodium C_(14/15) alcohol ethoxy-3-1.15 2.88 sulfate Sodium C_(14/1)5 alkyl sulphate 2.37 C_(14/15) alcoholethoxylate with average 1.17 1 7 moles of ethoxylation mono-C₈₋₁₀ alkylmono-hydroxyethyl 0.45 di-methyl quaternary ammonium chloride Di methylhydroxyl ethyl lauryl 0.18 ammonium chloride Zeolite A 13.9 4.7 0.01 2.91.8 Sodium Silicate 1.6. ratio 4 0.2 4 4 Sodium Silicate 2.35. ratio 8Citric Acid 2.5 1.4 Sodium tripolyphosphate 5 Sodium Carbonate 24.1 3016.9 24.4 21 Nonanoyloxybenzenesuplhonate 5.78 2.81 0.96Oxaziridinium-based bleach booster 0.03 0.017 Tetrasodium S,S,- 0.2ethylenediaminedisuccinate Diethylenetriamine penta (methylene 0.61 0.33phosphonic acid), heptasodium salt Hydroxyethane dimethylene 0.29 0.45phosphonic acid Ethylene diamine tetraacetate 0.27 MgSO4 0.47 0.59940.782 Sodium Percarbonate 7 4.4 15.9 19.1 Tetra Acetyl Ethylene Diamine3.3 4.6 Sodium Perborate Monohydrate 1.2 Carboxymethyl cellulose (e.g.Finnfix 0.1 0.17 1.69 0.23 BDA ex CPKelco) Sodium Acrylic acid/maleicacid co- 0.0236 3.8 2 2.5 polymer (70/30) Sodium polyacrylate (SokalanPA30 4 0.84 CL) Terephthalate polymer 0.23 Polyethylene glycol/vinylacetate 0.89 0.89 0.91 random graft co polymer Photobleach-zincphthalocyanine 0.005 0.001 0.002 tetrasulfonate C.I. FluorescentBrightener 260 0.11 0.15 0.04 0.23 0.15 C.I. Fluorescent Brightener 3510.1 (Tinopal ® CBS) Suds suppressor granule 0.25 0.07 0.04Hyrdophobically modified carboxy 0.019 0.028 methyl cellulose(Finnifix ® SH-1) Bentonite 8.35 Miscellaneous (Dyes, perfumes, processaids, moisture and sodium Balance Balance Balance Balance Balancesulphate)

Granular Laundry Detergent Compositions and Their Components. Thepresent amylase is separately added to these formulations. ComponentDetergent Composition Surfactants A B C D E F G H I J K L M N C₁₀Nonionic 0.1843 0.1142 0.2894 0.1885 0.1846 0.1885 0.1979 0.1979 0.19790.1979 C₁₆₋₁₇ Branched alkyl 3.53 3.53 3.53 sulfate C₁₂₋₁₄ alkylsulphate Sodium linear 8.98 8.98 8.98 13.58 14.75 12.94 15.69 9.01 8.429.51 8.92 8.92 11.5 11.5 alkylbenzenesulfonate with aliphatic chainlength C₁₁-C₁₂ Sodium C_(14/15) alcohol 1.28 1.28 1.28 1.62 1.62 1.1251.125 ethoxy-3-sulfate Sodium C_(14/15) alkyl 2.36 2.36 2.36 sulphateC_(12/14) alcohol ethoxylate 2.9 with average 7 moles of ethoxylationC_(12/14) alcohol ethoxylate 2.44 with average 3 moles of ethoxylationC_(14/15) alcohol ethoxylate 0.97 1.17 0.97 1 1 1.5 1.5 with average 7moles of ethoxylation mono-C₈₋₁₀ alkyl mono- 0.45 hydroxyethyl di-methylquaternary ammonium chloride Di methyl hydroxyl 0.1803 0.195 0.45 ethyllauryl ammonium chloride Zeolite A 15.31 15.31 15.31 4.47 2.01 0.39 1.832.58 0.59 1.63 1.63 2 2

Granular Laundry Detergent Compositions and Their Components. Thepresent amylase is separately added to these formulations. ComponentDetergent Composition Surfactants A B C D E F G H I J K L M N Bentonite8.35 Sodium Silicate 0.16 4.53 5.62 4.53 4.75 4.75 4.75 4.75 1.6.ratioSodium Silicate 3.72 3.72 3.72 8.41 10.1 0.06 0.06 2.0.ratio SodiumSilicate 7.05 2.35.ratio Citric Acid 0.0066 1.4 1.84 1 1.1 1.1 1.1 1.1Sodium 5.06 5.73 tripolyphosphate Sodium Carbonate 26.1 26.18 26.1 15.929 12.65 15.93 21 27.31 20.2 23.3 23.3 23.3 23.3 Nonanoyl oxybenzene5.78 5.78 5.78 1.17 1.86 1.73 suplhonate Oxaziridinium-based 0.037 0.0370.037 0.0168 0.0333 0.024 0.021 0.021 0.015 0.015 bleach boosterTetrasodium S,S,- 0.26 0.26 0.26 0.26 ethylene diaminedisuccinateDiethylenetriamine 0.62 0.62 0.62 0.327 0.3272 penta (methylenephosphonic acid), heptasodium salt Hydroxyethane 0.45 0.2911 0.45 0.470.47 0.47 0.47 dimethylene phosphonic acid Ethylene diamine 0.2701 0.280.1957 tetraacetate MgSO4 0.056 0.056 0.056 0.47 0.54 0.79 0.6494 0.7930.83 0.83 0.82 0.82 Sodium Percarbonate 7.06 7.06 3.64 19.1 15.85 22.519.35 19.35 19.35 19.35 Tetra Acetyl Ethylene 4.554 3.71 5.24 4.51 4.514.51 4.51 Diamine

Granular Laundry Detergent Compositions and Their Components. Thepresent amylase is separately added to these formulations. ComponentDetergent Composition Surfactants A B C D E F G Sodium Perborate 1.475.55 Monohydrate Carboxymethyl cellulose 0.38 0.38 0.38 0.173 0.62 0.21(e.g. Finnfix BDA ex CPKelco) Sodium Acrylic 3.79 3.78 3.79 3.64 0.42.61 acid/maleic acid co- polymer (70/30) Sodium polyacrylate 3.78 3.783.78 0.842 (Sokalan PA30 CL) Terephthalate polymer Polyethylene 0.890.55 1.4 glycol/vinyl acetate random graft co polymer Photobleach-zincphthalocyanine tetrasulfonate C.I. Fluorescent 0.1125 0.1125 0.11250.043 0.15 0.1174 0.048 Brightener 260 C.I. Fluorescent 0.0952 0.1049Brightener 351 (Tinopal ® CBS) Suds suppressor granule 0.015 0.015 0.0150.031 Hyrdophobically modified carboxy methyl cellulose (Finnifix ®SH-1) Bentonite Miscellaneous (Dyes, Balance Balance Balance BalanceBalance Balance Balance perfumes, process aids, moisture and sodiumsulphate) Component Detergent Composition Surfactants H I J K L M NSodium Perborate Monohydrate Carboxymethyl cellulose 0.23 1.07 0.26221.01 1.01 1.01 1.01 (e.g. Finnfix BDA ex CPKelco) Sodium Acrylic 2.5 21.75 1.84 1.84 1.84 1.84 acid/maleic acid co- polymer (70/30) Sodiumpolyacrylate 0.0055 0.011 0.008 0.007 0.007 0.005 0.005 (Sokalan PA30CL) Terephthalate polymer 0.231 0.179 0.179 0.179 0.179 Polyethylene0.911 0.8924 0.911 0.96 0.96 0.96 0.96 glycol/vinyl acetate random graftco polymer Photobleach-zinc phthalocyanine tetrasulfonate C.I.Fluorescent 0.1455 0.2252 0.1455 0.153 0.153 0.171 0.171 Brightener 260C.I. Fluorescent Brightener 351 (Tinopal ® CBS) Suds suppressor granule0.04 0.0658 0.04 0.042 0.042 0.042 0.042 Hyrdophobically modifiedcarboxy methyl cellulose (Finnifix ® SH-1) Bentonite Miscellaneous(Dyes, Balance Balance Balance Balance Balance Balance Balance perfumes,process aids, moisture and sodium sulphate)

Dishwashing Detergent Gel Compositions 1 2 3 4 5 Ingredients (wt %) (wt%) (wt %) (wt %) (wt %) Polytergent ® SLF-18 1 1.3 0.8 1 0.9 SodiumBenzoate (33% 0.61 0.61 0.61 0.6 0.6 active) Xanthan gum 1 0.8 1.2 1 1.1Sodium Sulphate 10 10 10 8 10 Perfume 0.03 0.05 0.03 0.06 0.1 SodiumSilicate 2 Citric Acid (50% active) 12.5 12 GLDA 7 8 Protease 1 (44 mg0.7 0.3 active/g 4-Formyl-Phenyl 0.05 BoronicAcid Protease 2 (10 mg/g) 20.6 encapsulated Protease 3 (48 mg 0.5 active/g) Protease 4 (123 mgactive/g) Ethanol 0.3 Potassium Hydroxide 14.6 14.6 14.6 14 (45% active)Calcium Chloride (25% 1.8 1.8 1.8 1.1 0.4 active) Dye 0.05 0.05 0.050.05 0.02 Proxcel GXL ™ (19% 0.05 0.05 0.05 0.05 0.05 active) Acusol ™8209 0.34 0.34 0.3 0.35 0.3 Acusol ™ 425N (50% 3 3 3.5 2.5 2 active)Amylases (25 mg/g 0.2 0.5 0.4 0.3 0.1 active) Water & other adjunctBalance Balance Balance Balance Balance ingredients to 100% to 100% to100% to 100% to 100%

Powder Automatic Dishwashing Compositions Ingredients Wt % Composition 1Nonionic surfactant 0.4-2.5%  Sodium metasilicate 0-20% Sodium disilicate 0-20% Sodium triphosphate 0-40% Sodium carbonate 0-20% Sodiumperborate  2-9% Tetraacetyl ethylene diamine (TAED)  1-4% Sodium sulfate5-33% Enzymes 0.0001-0.1%    Composition 2 Nonionic surfactant (e.g.alcohol ethoxylate)  1-2% Sodium di silicate 2-30% Sodium carbonate10-50%  Sodium phosphonate  0-5% Trisodium citrate dehydrate 9-30%Nitrilotrisodium acetate (NTA) 0-20% Sodium perborate monohydrate 5-10%Tetraacetyl ethylene diamine (TAED)  1-2% Polyacrylate polymer (e.g.maleic acid/acrylic 6-25% acid copolymer) Enzymes 0.0001-0.1%    Perfume0.1-0.5%  Water 5--10 Composition 3 Nonionic surfactant 0.5-2.0%  Sodiumdi silicate 25-40%  Sodium citrate 30-55%  Sodium carbonate 0-29% Sodiumbicarbonate 0-20% Sodium perborate monohydrate 0-15% Tetraacetylethylene diamine (TAED)  0-6% Maleic acid/acrylic acid copolymer  0-5%Clay  1-3% Polyamino acids 0-20% Sodium polyacrylate  0-8% Enzymes0.0001-0.1%   

Powder Automatic Dishwashing Compositions Ingredients Wt % Composition 4Nonionic surfactant  1-2% Zeolite MAP 0-42% Sodium disilicate 0-34%Sodium citrate 0-12% Sodium carbonate 0-20% Sodium perborate monohydrate7-15% Tetraacetyl ethylene diamine (TAED)  0-3% Polymer  0-4% Maleicacid/acrylic acid copolymer  0-5% Organic phosphonate  0-4% Clay  1-2%Enzymes 0.0001-0.1%    Sodium sulfate Balance Composition 5 Nonionicsurfactant  1-7% Sodium disilicate 18-30%  Trisodium citrate 10-24% Sodium carbonate 12-20%  Monopersulfate (2 KHSOsoKHS04 °K2S04) 15-21% Bleach stabilizer 0.1-2%  Maleic acid/acrylic acid copolymer  0-6%Diethylene triarnine pentaacetate, pentasodium salt 0-2.5%  Enzymes0.0001-0.1%    Sodium sulfate, water Balance

Powder and Liquid Dishwashing Composition with Cleaning SurfactantSystem Ingredients Wt % Nonionic surfactant 0-1.5%   Octadecyldimethylamine N-oxide 0-5% dihydrate 80:20 wt C18/C16 blend of octadecyl0-4% dimethylamine N-oxide dihydrate and hexadecyldimethyl amine Noxidedehydrate 70:30 wt C18/C16 blend ofoctadecyl 0-5% bis(hydroxyethyl)amine N-oxide anhydrous and hexadecyl bis (hydroxyethyl)amine N-oxide anhydrous C13-C1S alkyl ethoxysulfate with an 0-10% average degree of ethoxylation of 3 C12-C1S alkyl ethoxysulfate with an0-5% average degree of ethoxylation of 3 C13-C1S ethoxylated alcoholwith an 0-5% average degree of ethoxylation of 12 A blend of C 12-C ISethoxylated 0-6.5%   alcohols with an average degree of ethoxylation of9 A blend of C 13-C IS ethoxylated 0-4% alcohols with an average degreeof ethoxylation of 30 Sodium disilicate 0-33%  Sodium tripolyphosphate0-46%  Sodium citrate 0-28%  Citric acid 0-29%  Sodium carbonate 0-20% Sodium perborate monohydrate 0-11.5%   Tetraacetyl ethylene diamine(TAED) 0-4% Maleic acid/acrylic acid copolymer 0-7.5%   Sodium sulfate0-12.5%   Enzymes 0.0001-0.1%   

Non-Aqueous Liquid Automatic Dishwashing Composition Ingredients Wt %Liquid nonionic surfactant (e.g. 2.0-10.0% alcohol ethoxylates) Alkalimetal silicate 3.0-15.0% Alkali metal phosphate   0-40.0% Liquid carrierselected from 25.0-45.0%  higher glycols, polyglycols, polyoxides,glycol ethers Stabilizer (e.g. a partial ester of  0.5-7.0% phosphoricacid and a C16-C18 alkanol) Foam suppressor (e.g. silicone)   0-1.5%Enzymes 0.0001-0.1% 

Non-Aqueous Liquid Dishwashing Composition Ingredients Wt % Liquidnonionic surfactant 2.0-10.0% (e.g. alcohol ethoxylates) Sodium silicate3.0-15.0% Alkali metal carbonate 7.0-20.0% Sodium citrate  0.0-1.5%Stabilizing system (e.g.  0.5-7.0% mixtures of finely divided siliconeand low molecular weight dialkyl polyglycol ethers) Low molecule weight5.0-15.0% polyacrylate polymer Clay gel thickener (e.g. 0.0-10.0%bentonite) Hydroxypropyl cellulose  0.0-0.6% polymer Enzymes0.0001-0.1%  Liquid carrier selected from Balance higher lycols,polyglycols, polyoxides and glycol ethers

Thixotropic Liquid Automatic Dishwashing Composition Ingredients Wt % C12-C 14 fatty acid 0-0.5% Block co-polymer surfactant 1.5-15.0%   Sodiumcitrate  0-12% Sodium tripolyphosphate  0-15% Sodium carbonate   0-8%Aluminium tristearate 0-0.1% Sodium cumene sulfonate 0-1.7% Polyacrylatethickener 1.32-2.5%   Sodium polyacrylate 2.4-6.0%   Boric acid 0-4.0%Sodium formate 0-0.45%  Calcium formate 0-0.2% Sodium n-decydiphenyloxide 0-4.0% disulfonate Monoethanol amine (MEA) 0-1.86%  Sodiumhydroxide (50%) 1.9-9.3%   1,2-Propanediol 0-9.4% Enzymes 0.0001-0.1%   Suds suppressor, dye, Balance perfumes, water

Liquid Automatic Dishwashing Composition Ingredients Wt % Alcohol 0-20%ethoxylate Fatty acid ester 0-30% sulfonate Sodium 0-20% dodecyl sulfateAlkyl 0-21% polyglyco side Oleic acid 0-10% Sodium 0-33% disilicatemonohydrate Sodium citrate 0-33% dihydrate Sodium stearate 0-2.5% Sodium 0-13% perborate monohydrate Tetraacetyl  0-8% ethylene diamine(TAED) Maleic  4-8% acid/acrylic acid copolymer Enzymes 0.0001-0.1%   

Liquid Automatic Dishwashing Composition Containing Protected BleachParticles Ingredients Wt % Sodium silicate 5-10% Tetrapotassium 0-25%pyrophosphate Sodium  0-2% triphosphate Potassium carbonate  4-8%Protected bleach 5-10% particles, e.g. chlorine Polymeric thickener0.7-1.5%  Potassium  0-2% hydroxide Enzymes 0.0001-0.1%    Water Balance

Composition Composition of Model of Model Detergent A: Detergent B:Amount % active Amount % active Compound g/100 g ingredient g/100 gingredient Surfactants Na-LAS (92%) 10.87 10 10.87 10 (NacconoI90G)(anionic) (linear alkylbenzene sulfonate) STEOL CS-370E (70%) 7.14 57.14 5 (anionic), CH3(CH2)m- (OCH2CH2)3—OS03—, where m~11-13 Bio-softN25-7 (99.5%) 5 5 5 5 (non-ionic),: CH3(CH2)m- (OCH2CH2h—OH, where andm~11-14 Oleic acid (fatty acid) 2 2 2 2 Solvents H20 62 65 62 65 Ethanol0.5 0.5 0.5 0.5 STS (sodium p-toluene 3.75 1.5 3.75 1.5 sulfonate (40% 

Mono propylene glycol 2 2 2 2 Builder Tri-sodium-citrate 4 4 0 0Diethylene triamine penta 0 0 1.5 1.5 acetic acid (DTPA) Triethanolamine(TEA) 0.5 0.5 0.5 0.5 Stabilizer Boric Acid 1.5 1.5 1.5 1.5 Minors 10NNaOH (for adjustment 0.8 0.8 0.8 0.8 to pH 8.5)

Liquid Detergent and Cleaning Agent Compositions Ingredients E1 E2 E3 C1C2 C3 C4 C5 Gellan gum 0.2 0.2 0.15 0.15 Xanthan gum 0.15 0.15 0.5 0.2Polyacrylate (Carbopol 0.4 0.4 0.6 0.6 Aqua 30) C₁₂₋₁₄-fatly alcoholwith 7 22 10 10 10 10 10 10 10 EO C₉₋₁₃- 10 10 10 10 10 10 10alkylbenzenesulfonate, Na salt C₁₂₋₁₄-alkylpolyglycoside 1 Citric acid1.6 3 3 3 3 3 3 3 Dequest ® 2010 0.5 1 1 1 1 1 1 1Hydroxyethylidene-1,1- diphosphonic acid, tetrasodium salt (fromSolutia) Sodium lauryl ether 10 5 5 5 5 5 5 5 sulfate with 2 EOMonoethanolarnine 3 3 3 3 3 3 3 3 C₁₂₋₁₈-fatty acid 7.5 7.5 7.5 7.5 7.57.5 7.5 7.5 Propylene glycol 6.5 6.5 6.5 6.5 6.5 6.5 6.5 Sodium cumenesulfonate 2 2 2 2 2 2 2 Enzymes, dyes, + + + + + + + + stabilizersMicrocapsules with about 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 2000 μmdiameter Water To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100Flow limit (Pas) 0.58 1.16 1.16 no no no yes no

All purpose Alkaline detergent Compositions (all-purpose. glass.kitchen) Hard surface cleaning detergent composition Composition [% bywt.] E1 E2 E3 E4 Fatty alcohol ethoxylate 1 3 5 0.5 C12-7EOAlkylbenzenesulfonic acid Na 3 1 2 4 salt Octyl sulfate 3 2 2 2 Sodiumcarbonate 1.5 0.5 1.0 1.5 Citric acid 0.5 0.5 0.5 0.5 Fatty acid 0.5 0.50.5 1.0 Ethanol 5 3 5 3 Perfume 0.2 0.2 0.2 0.2 Water To 100 To 100 To100 To 100

Acidic Detergent Compositions (bath, toilet) Composition [% by wt.] E5E6 E7 E8 Fatty alcohol ether sulfate 2 3 5 2 C12-2EO sodium salt Ethanol3 3 3 3 Citric acid 3 10 3 10 Thickener xanthan Kelzan ASX-T 0.05 0.05Perfume 0.1 0.1 0.1 0.1 Water To 100 To 100 To 100 To 100

Cleaning Paste Composition Composition [% by wt.] E9 C 12 Fatty alcoholsulfate 20 C16-18 Fatty alcohol ethoxylate 25 EO 20 C 12-18 Fatty acid10 monoethanolamide Sodium sulfate 40 Sodium carbonate 5 Cellulose 4.899Dye 0.001 Perfume 0.1

Self Foaming Cleaning Powder Composition Composition [% by wt.] E10 C 12Fatty alcohol sulfate 2 Sodium sulfate 37.899 Sodium carbonate 25 CitricAcid 35 Dye 0.001 Perfume 0.1

Compositions of a Clear Aqueous Detergent and Cleaning Agent having aflow limit Ingredients V1 E1 E2 E3 E4 E5 1,2 Propane diol 8 0 2 6 4 2Dipropylene glycol 0 8 6 2 4 2 Polyacrylate (Carbopol 3 3 3 3 3 Aqua 30)Polyacrylate (Polygel — — — — — 1.8 W301) C₁₂₋₁₄-fatty alcohol with 7 1010 10 10 10 10 EO C₉₋₁₃- 10 10 10 10 10 — alkylbenzenesulfonate, Na saltCitric Acid 3 3 3 3 3 2 Dequest ® 2010 1 1 1 1 1 —Hydroxyethylidene-1,1- diphosphonic acid, tetrasodium salt (ex Solutia)Dequest ® 2066 — — — — — 0.7 Diethylene triamine penta(methylenephosphonic acid) hepta Na salt (ex Solutia) Sodium laurylether 10 10 10 10 10 5 sulfate with 2 EO Monoethanolamine 3 3 3 3 3 2C₁₂₋₁₈-fatty acid Na salt 5.5 5.5 5.5 5.5 5.5 5.5 Enzymes, dyes,stabilizers + + + + + + Microcapsules with about 0.5 0.5 0.5 0.5 0.5 0.52000 μm diameter Water To 100 To 100 To 100 To 100 To 100 To 100 Flowlimit (Pas) 0.4 0.6 0.6 0.8 1.0 0.6 Appearance Cloudy Clear Clear ClearClear Clear

Liquid Laundry Detergent Ingredients Wt % ABS (alkyl benzenesulphonate)10 FAEOS 5 C_(12/14) 7EO 10 C_(12/18) Fatty Acid 5 Glycerol 5 Sodiumcitrate 3 Protease/Amylase/Cellulase 1 Tinopal ® DMS-X (opticalbrightener 0.2 manufactured by Ciba) Water To 100

Granular Laundry Detergent Ingredients Wt % ABS (alkylbenzenesulphonate) 11 C_(13/15) 7EO 3 Sodium carbonate 20 Sodiumhydrogencarbonate 5 Sodium sulphate 25 Sodium silicate 5 Sodiumpercarbonate 13 TAED 5 Sodium polyacrylate 4.5 Enzymes (protease,amylase, and 3.5 cellulose) Water To 100

Aqueous Liquid Washing Product Formulations (without- FWM1 and with-FWM20.5% hyperbranched polyesteramide Formulation FWM1 FWM2 C₁₂₋₁₄-fattyalcohol with 2 EO 5 5 LAS 10 10 C₁₂₋₁₈-fatty alcohol with 7 EO 10 10C₁₂₋₁₈ soap 8 8 Citrate 4 4 1,2-propanediol 5 5 Hybrane ® SIP 2100(manufactured by 0.5 DSM)

Liquid Laundry Detergent Compositions Wt % Detergent Composition E1 E2E3 C₁₂₋₁₄ fatty alcohol with 7 EO 5 4 10 C₉₋₁₃ alkylbenzene sulfonate,Na salt 10 10 10 Sodium lauryl ether sulfate with 2 EO — — 8 Activesubstance (specific 1 1 1 polycarbonate-, polyurethane-, and/orpolyureapolyorganosiloxane compounds or precursor compounds thereof ofthe reactive cyclic carbonate and urea type Polyacrylate thickener — — 1Sodium percarbonate 15 18 — TAED 3 3 — C₁₂₋₁₈ fatty acid, Na salt 1 1.57.5 PVA/Maleic acid copolymer 4.5 2 — Citric acid, Na salt 2.5 — 2Phosphonic acid, Na salt 0.5 0.5 1 Sodium carbonate 10 20 — Propane diol— — 6.5 Zeolite A 25 25 — Boric Acid Sodium salt — — 1.2 Siliconedefoamer 2.5 1.3 0.1 Enzymes (protease, amylase, cellulase) + + +Colorant + + + Perfume 0.5 0.2 0.8 Water — — To 100 Sodium sulfate — To100 — Sodium bicarbonate To 100 — —

Example formulations of preferred phosphate-free automatic dishwashingagents Formulation Formulation Formulation Formulation 1 2 3 4Ingredient (wt %) (wt %) (wt %) (wt %) Citrate  5 to 60 10 to 55 15 to50 15 to 50 Sodium  1 to 20  2 to 15  4 to 10  4 to 10 percarbonateBleach catalyst 0.01 to 3   0.02 to 2   0.02 to 2   0.02 to 1  Copolymer¹ 0.1 to 30  0.5 to 25  1.0 to 20  1.0 to 20  Nonionic  1 to 102 to 8 2 to 8 3 to 6 surfactant² Misc To 100 To 100 To 100 To 100

Example formulations of preferred phosphate-free automatic dishwashingagents Formulation Formulation Formulation Formulation 5 6 7 8Ingredient (wt %) (wt %) (wt %) (wt %) Citrate  5 to 60 10 to 55 15 to50 15 to 50 Sodium  1 to 20  2 to 15  4 to 10  4 to 10 percarbonatePhosphonate 2 to 8 2 to 8 2 to 8 2 to 8 Copolymer¹ 0.1 to 30  0.5 to 25 1.0 to 20  1.0 to 20  Nonionic  1 to 10 2 to 8 2 to 8 3 to 6 surfactant²Misc To 100 To 100 To 100 To 100

Example formulations of preferred phosphate-free automatic dishwashingagents Formulation Formulation Formulation Formulation 9 10 11 12Ingredient (wt %) (wt %) (wt %) (wt %) Citrate  5 to 60 10 to 55 15 to50 15 to 50 Sodium  1 to 20  2 to 15  4 to 10  4 to 10 percarbonateEnzyme 0.1 to 6   0.2 to 5   0.4 to 5   0.4 to 5   Copolymer¹ 0.1 to 30 0.5 to 25  1.0 to 20  1.0 to 20  Nonionic  1 to 10 2 to 8 2 to 8 3 to 6surfactant² Misc To 100 To 100 To 100 To 100

Example formulations of preferred phosphate-free automatic dishwashingagents Formulation Formulation Formulation Formulation 13 14 15 16Ingredient (wt %) (wt %) (wt %) (wt %) Citrate  5 to 60 10 to 55 15 to50 15 to 50 Carbonate/  2 to 40  2 to 40  2 to 40  2 to 40 hydrogencarbonate Silicate  0 to 15  0 to 15  0 to 15 0.1 to 10   Phosphonate  0to 14  0 to 14  0 to 14 2 to 8 Sodium  1 to 20  2 to 15  4 to 10  4 to10 percarbonate Bleach catalyst 0.01 to 3   0.02 to 2   0.02 to 2   0.02to 1   Copolymer¹ 0.1 to 30  0.5 to 25  1.0 to 20  1.0 to 20  Nonionic 1 to 10 2 to 8 2 to 8 3 to 6 surfactant² Enzyme 0.1 to 6   0.2 to 5  0.4 to 5   0.4 to 5   Misc To 100 To 100 To 100 To 100 ¹Copolymercomprising i) monomers from the group of mono- or polyunsaturatedcarboxylic acids ii) monomers of the general formula R¹(R²)C═C(R³)—X—R⁴,in which R¹ to R³ mutually independently denote —H, —CH₃ or —C₂H₅, Xdenotes an optionally present spacer group which is selected from —CH₂—,—C(O)O— and —C(O)—NH—, and R⁴ denotes a straight chain or branchedsaturated alkyl residue with 2 to 22 carbon atoms or denotes anunsaturated, preferably aromatic residue with 6 to 22 carbon atoms iii)optionally further monomers ²Nonionic surfactant of the general formulaR¹—CH(OH)CH₂0—(AO)w—(A′0)_(x)—(A″0)_(y)—(A″′0)_(z)—R₂, in which R¹denotes a straight-chain or branched, saturated or mono- orpolyunsaturated C6-24 alkyl or alkenyl residue; R² denotes a linear orbranched hydrocarbon residue with 2 to 26 carbon atoms; A, A′, A″ andA″′ mutually independently denote a residue from the group comprising—CH₂CH₂, —CH₂CH₂—CH₂, —CH₂CH₂—CH(CH₃), CH₂—CH₂—CH₂CH₂,—CH₂—CH—(CH₃)—CH₂—, —CH₂—CH(CH₂ —CH₃), w, x, y and z denote valuesbetween 0.5 and 120, wherein x, y and/or z may also be 0.

Composition of phosphate-free automatic dishwashing detergents Rawmaterial V1 E1 Citrate 23 23 MGDA 8 8 Copolymer¹ 12 12 HEDP 2 2 Soda 2828 Sodium percarbonate 10 10 TAED 2.4 2.4 Protease 2 2 Amylase 1.8 1.8Non-ionic surfactant² 5 — Non-ionic surfactant³ — 5 Misc To 100 To 100

Textile Washing Agent wt % pure Ingredient substance Xanthan 0.3-0.5Anti foaming agent 0.2-0.4 Glycerol 6-7 Ethanol 0.3-0.5 FAEOS 4-7 Nonionic surfactant 24-28 (FAEO, APG among others) Boric acid 1 Sodiumcitrate dihydrate 1-2 Soda 2-4 Coconut fatty acids 14-16 HEDP 0.5 PVP  0-0.4 Optical brightener   0-0.05 Dye    0-0.001 Perfume 0-2 Waterdemineralized remainder

Example detergent compositions for application to a substrate WeightPercent (actives %) Ingredients D1 D2 D3 D4 D5 Sodium dodecyl benzene26.09 17.30 15.60 17.70 16.70 sulfonate Sodium alkyl C₁₄₋₁₅/7EO 13.80 —— — — ether sulfate Linear alcohol ethoxylate 13.44 5.4 14.6 5.5 5.2C₁₄₋₁₅/7EO Polyethylene glycol PEG 75 2 1.4 1.3 1.4 1.4 Polyoxyethylene(100) 21.99 15.6 14.1 15.9 15.1 stearyl ether Sodium silicate SiO₂/Na₂O3.72 16.6 15 17 16 ratio 1.6-1.8 Sodium Silicate 7 — — — — (Britesil ®C24) Sodium Carbonate — 6.5 5.9 6.7 6.3 Sodium tetraborate — 11.9 10.812.2 11.5 decahydrate Sodium polyacrylate — 1.8 1.7 — 5.2 ~4500 MWEDTA-tetrasodium salt — 0.1 0.1 0.1 0.1 Optical brightener 0.15 0.1 0.090.1 0.1 (Tinopal ® CBS-X) Dyes and fragrances 0.9 0.9 0.81 1.01 0.91Water 10.92 22.10 19.90 22.4 21.5

Example fabric conditioning compositions for application to a substrateWeight Percent (actives %) Ingredients FS1 FS2 FS3 FS4 FS5Di-(hydrogenated tallow) dimethyl 33.6 33.2 44.4 22.2 33.2 ammoniummethyl sulfate Unsaturated trialkylglycerides 16.8 16.6 22.2 11.1 16.6Hydrogenated tallow fatty acid 16.8 16.6 22.2 11.1 16.6 C₁₂₋₁₈ cocofatty acid 11.2 11.1 — 11.1 — C₁₂₋₁₈ fatty alcohol ethoxylate (7EO) 11.211.1 — — 16.6 Fragrance oil 10.4 11.4 11.2 11.2 17

Exemplary Automatic Dishwashing Agents Wt % Ingredient Formula 1 Formula2 Formula 3 Formula 4 Citrate 12-50 15-40 12-50 15-40 Dicarboxylic acid 1-18  1-18  2-16  4-12 Phosphate — — — — Bleaching Agent — — — — MiscTo 100 To 100 To 100 To 100

Additional Exemplary Automatic Dishwashing Agents Wt % IngredientFormula 1 Formula 2 Formula 3 Formula 4 Citrate 12-50 15-40 12-50 15-40Dicarboxylic acid  1-18  1-18  2-16  4-16 Carbonate  5-50 10-40  5-5010-40 Phosphate — — — — Bleaching Agent — — — — Misc To 100 To 100 To100 To 100

Additional Exemplary Automatic Dishwashing Agents Wt % IngredientFormula 1 Formula 2 Formula 3 Formula 4 Citrate 12-50 15-40 12-50 15-40Dicarboxylic acid  1-18  1-18  2-16  4-12 Carbonate  5-50 10-30  5-5010-30 Phosphonate 1-8 1-8 1.2-6   1.2-6   Phosphate — — — — BleachingAgent — — — — Misc To 100 To 100 To 100 To 100

Preferred Automatic Dishwashing Agents Wt % Ingredient Formula 1 Formula2 Formula 3 Formula 4 Citrate 12-50 15-40 12-50 15-40 Dicarboxylic acid 1-18  1-18  2-16  4-12 Carbonate  0-50  0-30  0-30  0-30 Phosphonate0-8 0-8 0-8 0-8 Phosphate — — — — Bleaching Agent — — — — Misc To 100 To100 To 100 To 100

Additional Preferred Automatic Dishwashing Agents Wt % IngredientFormula 1 Formula 2 Formula 3 Formula 4 Citrate 12-50 15-40 12-50 15-40Maleic acid  1-18  1-18  2-16  4-12 Carbonate  5-50 10-30  5-50 10-30Phosphonate 1-8 1-8 1.2-6   1.2-6   Phosphate — — — — Bleaching Agent —— — — Misc To 100 To 100 To 100 To 100

Preferred Automatic Dishwashing Agents Wt % Ingredient Formula 1 Formula2 Formula 3 Formula 4 Citrate 12-50 15-40 12-50 15-40 Dicarboxylic acid 1-18  1-18  2-16  4-12 Carbonate  0-50  0-30  0-30  0-30 Phosphonate0-8 0-8 0-8 0-8 Non-ionic 0.1-15  0.1-15  0.5-8   0.5-8   surfactantPhosphate — — — — Bleaching Agent — — — — Misc To 100 To 100 To 100 To100

Additional Preferred Automatic Dishwashing Agents Wt % IngredientFormula 1 Formula 2 Formula 3 Formula 4 Citrate 12-50 15-40 12-50 15-40Maleic acid  1-18  1-18  2-16  4-12 Carbonate  5-50 10-30  5-50 10-30Phosphonate 1-8 1-8 1.2-6   1.2-6   Non-ionic 0.1-15  0.1-15  0.5-8  0.5-8   surfactant Phosphate — — — — Bleaching Agent — — — — Misc To 100To 100 To 100 To 100

Preferred Automatic Dishwashing Agents Wt % Ingredient Formula 1 Formula2 Formula 3 Formula 4 Citrate 12-50 15-40 12-50 15-40 Dicarboxylic acid 1-18  1-18  2-16  4-12 Carbonate  0-50  0-30  0-30  0-30 Phosphonate0-8 0-8 0-8 0-8 Sulfo copolymer  0-20  0-20  0-20  0-20 Non-ionic  0-15 0-15 0-8 0-8 surfactant Enzyme 0.1-12  0.1-12  0.5-8   0.5-8  preparations Phosphate — — — — Bleaching Agent — — — — Misc To 100 To100 To 100 To 100

Additional Preferred Automatic Dishwashing Agents Wt % IngredientFormula 1 Formula 2 Formula 3 Formula 4 Citrate 12-50 15-40 12-50 15-40Maleic acid  1-18  1-18  2-16  4-12 Carbonate  5-50 10-30  5-50 10-30Phosphonate 1-8 1-8 1.2-6   1.2-6   Sulfo copolymer  0-20  0-20  0-20 0-20 Non-ionic 0.1-15  0.1-15  0.5-8   0.5-8   surfactant Enzyme0.1-12  0.1-12  0.5-8   0.5-8   preparations Phosphate — — — — BleachingAgent — — — — Misc To 100 To 100 To 100 To 100

Preferred Automatic Dishwashing Agents Wt % Ingredient Formula 1 Formula2 Formula 3 Formula 4 Citrate 12-50 15-40 12-50 15-40 Dicarboxylic acid 1-18  1-18  2-16  4-12 Carbonate  0-50  0-30  0-30  0-30 Phosphonate0-8 0-8 0-8 0-8 Sulfo copolymer  0-20  0-20  0-20  0-20 Non-ionic  0-15 0-15 0-8 0-8 surfactant Enzyme  0-12  0-12 0-8 0-8 preparations OrganicSolvent 0.1-15  0.5-8   0.1-15  0.5-8   Phosphate — — — — BleachingAgent — — — — Misc To 100 To 100 To 100 To 100

Additional Preferred Automatic Dishwashing Agents Wt % IngredientFormula 1 Formula 2 Formula 3 Formula 4 Citrate 12-50 15-40 12-50 15-40Dicarboxylic acid  1-18  1-18  2-16  4-12 Carbonate  5-50 10-30  5-5010-30 Phosphonate 1-8 1-8 1.2-6   1.2-6   Sulfo copolymer  0-20  0-20 0-20  0-20 Non-ionic 0.1-15  0.1-15  0.5-8   0.5-8   surfactant Enzyme0.1-12  0.1-12  0.5-8   0.5-8   preparations Organic Solvent 0.1-15 0.5-8   0.1-15  0.5-8   Phosphate — — — — Bleaching Agent — — — — MiscTo 100 To 100 To 100 To 100

Automatic Dishwashing Agents Wt % Ingredient C 1 E 1 Sodium citrate 9 9Potassium hydroxide 7 7 Sodium carbonate 14 14 Maleic acid — 1 Sulfopolymer 4.2 4.2 HEDP 1.5 1.5 Non-ionic surfactant 2 2 Proteasepreparation 2 2 Amylase preparation 0.8 0.8 Alkanolamine 1.5 1.5Thickener 2 2 Water, misc To 100 To 100

Manual Dishwashing Agents Wt % Ingredient Invention 1 Invention 2Invention 3 Invention 4 Invention 5 Invention 6 Invention 7 Fattyalcohol ether 10 13.33 12 12 13.3 13.3 13.3 sulfateCocamidopropylbetaine 2.5 3.33 3.1 3.1 3 3 3 Sce. Alkanesulfonate 2.53.33 2.9 2.9 3.7 3.7 3.7 Fatty alcohol 9 6 — — — — — ethoxylate Sodiumchloride 24 24 22 24 20 24 20 Ethanol — — 2 2 2.5 2.5 4 Perfume 0.2 0.30.3 0.3 0.3 0.3 0.3 Colorant 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Water 51.6049.51 57.5 55.5 57 53 55.5

Antibacterially active detergent/cleaning agent Ingredient V1 E1 E2 E3E4 E5 C₁₂₋₁₈ fatty alcohol with 7EO 12 12 12 5 5 — N-cocoalkyl N,Ndimethylamine 1.95 1.95 1.95 2 2 — oxide Esterquat (N-methyl-N-(2 — — —— — 15 hydroxyethyl)-N-N- (ditallowacyloxyethyl)ammonium methosulfateAgNO₃•H₂O 0.0043 0.0043 0.0043 0.004 0.004 0.004 C14 fatty acid 5 5 — —— — Farnesol 0.02 0.02 0.02 0.02 0.02 0.02 Coco Fatty acid 2.5 2.5 2.512 — — Citric Acid — — — 1.0 0.1 — H₂O₂ — 0.5 0.035 2 5 0.5 NaOH 0.350.35 0.35 1.9 — — NH₄OH 0.04 0.04 0.04 0.06 — — 2-Propanol — — — — —1.67 MgCl₂ × 6H₂O — — — — — 0.01 Perfume A 1.00 1.00 1.00 1.00 1.00 0.75Water To 100 To 100 To 100 To 100 To 100 To 100 pH 8.5 8.5 8.5 8.5 5.52.6

Detergent containing anti-grey agent Ingredients M1 (wt %) C₉₋₁₃alkylbenzenesulfonate sodium salt 10 Sodium lauryl ether sulfate with2EO  5 C₁₂₋₁₈ fatty alcohol with 7EO 10 C₁₂₋₁₄ alkyl polyglycoside  2C₁₂₋₁₈ fatty acid sodium salt  8 Glycerol  5 Trisodium citrate  1Polyacrylate  2 Active ingredient (anti-grey agent-a polycarbonate-,  1polyurethane-, and/or polyurea-polyorganosiloxane compound or aprecursor compound use in the production thereof) Enzyme, dye, opticalbrightener + Water To 100

Example detergent compositions for application to a substrate WeightPercent (actives %) Ingredients D1 D2 D3 D4 D5 Sodium dodecyl benzene26.09 17.30 15.60 17.70 27.00 sulfonate Sodium alkyl C₁₄₋₁₅/7EO 13.8014.00 ether sulfate Linear alcohol ethoxylate 13.44 5.40 14.60 5.5014.00 C₁₄₋₁₅/7EO Linear alcohol ethoxylate 23.00 C₁₂₋₂₀/7EO PolyethyleneGlycol PEG-75 2.00 1.40 1.30 1.40 2.00 Polyoxyethylene (100) 21.99 15.6014.10 15.90 stearyl ether Sodium Silicate Si0₂/Na₂0 3.72 16.60 15.0017.00 ratio 1.6-1.8 Sodium Silicate 7.00 11.00 (Britesil ® C24) SodiumCarbonate 6.50 5.90 6.70 Sodium tetraborate 11.90 10.80 12.20decahydrate Sodium polyacrylate 1.80 1.70 −4,500 MW EDTA-tetrasodiumsalt 0.10 0.10 0.10 Optical brightener 0.15 0.10 0.09 0.10 0.20(Tinopal ® CBS-X) Dyes and fragrances 0.90 0.90 0.81 1.01 0.35 Water10.92 22.10 19.90 22.40 9.55

Example enzyme containing compositions for application to a substrateWeight Percent (actives %) Ingredients E1 E2 E3 E4 E5 PolyethyleneGlycol 98.60 99.10 PEG-75 Fatty acid based matrix 1 98.9 99.10 Fattyacid based matrix 2 98.80 Protease 0.10 0.10 0.12 0.10 0.10 Mannanase0.02 0.02 0.02 Amylase 0.12 0.25 0.1 0.12 0.25 Cellulase 0.08 0.1 0.08Lipase 0.08 0.08 Pectate Lyase 0.05 Enzyme Stabilizers 1.00 0.55 0.750.75 0.55 Fatty acid based matrix 1 is comprised of 20 wt. % of thesodium salt of coconut fatty acid, 50 wt. % of non polymeric polyols(sorbitol, glycerin, propylene glycol, sucrose and glucose), 15 wt. % ofanionic and nonionic surfactants, and 15 wt. % of water. Fatty acidbased matrix 2 is comprised of 20 wt. % of the sodium salt of stearicacid, 3 wt. % of the sodium salt of lauric acid, 3 wt. % of the sodiumsalt of myristic acid, 50 wt. % of non polymeric polyols (sorbitol,glycerin, and propylene glycol), 2 wt. % of lauric acid, 2 wt. % ofstearic acid, 10 wt. % of anionic surfactant, and 10 wt. % of water.

Detergent Composition (% by Ingredients weight) Soap (saturated C₁₂₋₂₄fatty acid soaps 5.42 and oleic acid soap) Sodium C₁₂₋₁₄ alkylbenzenesulfonate 22.67 Sodium C₁₄₋₁₆ fatty alcohol sulfate 4.59 C₁₂₋₁₈fatty alcohol.5EO 0.81 Sodium carbonate 4.55 Zeolite A 29.86 Sodiumsilicate 8.00 Acrylic acid/maleic acid copolymer 16.16 Opt. brightener0.45 Phosphonate 2.30 NaOH, 50% 0.63 Water 3.88 Other salts 0.68

Detergent Composition Detergent composition 59.5% Coated bleaching agent23.3% (Na percarbonate) Coated bleach activator (TAED)   7% Citric acidmonohydrate 10.2%

Particulate detergent composition Ingredient % wt sodiumdodecylbenzenesulphonate 8.5 c12-C15 primary alcohol, condensed 4 with 7moles of ethylene oxide sodium-hardened rapeseed oil soap 1.5 sodiumtriphosphate 33 sodium carbonate 5 sodium silicate 6 sodium sulphate 20water 9 fluorescers, soil-suspending agents, dyes, perfumes minoramounts sodium perborate 12 tetraacetyl ethylene diamine (TAED)(granules) 2 proteolytic enzyme (Savinase ex. Novo) 0.4

Detergent composition A 9% anionic detergent 1% nonionic detergent 21.5%sodium tripolyphosphate 7% sodium perborate 0.6% Savinase (a proteolyticenzyme) balance sodium sulphate + minor ingredients

Detergent composition B 9% anionic detergent 4% nonionic detergent 28%zeolite 4.5% nitrilotriacetate 5.5% sodium perborate 3.5%tetraacetylethylenediamine 0.5% Savinase balance sodium sulphate + minoringredients

Detergent composition C 5% anionic detergent 4% nonionic detergent 1%soap 30% zeolite 3.% copolymer of acrylic acid with mateic anhydride7.5% sodium perborate 3% tetraacetylethylenediamine balance sodiumsulphate + minor ingredients

Detergent composition D 8% anionic synthetic detergent 4% nonionicsynthetic detergent 4% soap 35.% sodium carbonate 20% powdered calcite6% sodium perborate 2% tetraacetylethylenediamine 0.5% Savinase balancesodium sulphate + minor ingredients

Laundry detergent composition Ingredients Parts by weight Sodium dodecylbenzene sulphonate 8.5 C12-C15 primary alcohol, condensed 4 with 7 molesof ethylene oxide Sodium-hardened rapeseed oil soap 1.5 Sodiumtriphosphate 33 Sodium carbonate 5 Sodium silicate 6 Sodium sulphate 20Water 9 Fluorescers, soil-suspending agents, minor amount dyes, perfumesSodium perborate 12 Tetraacetyl ethylene diamine (TAED) 2 (granules)Proteolytic enzyme (Savinase ex NOVO) 0.4

Laundry detergent compositions A B C D sodium 9 9 9 9 dodecylbenzenesulphonate C13-C15 linear 1 4 4 1 primary alcohol, condensed with 7moles of ethylene oxide (e.g. Synperonic A7) C13-C15 linear 3 0 0 3primary alcohol, condensed with 3 moles of ethylene oxide (e.g.Synperonic A3) sodium 23 23 0 0 tripolyphosphate zeolite type 4A 0 0 2424 copolymer of acrylic 4 4 acid with maleic anhydride sodiumpolyacrylate 2 2 0 0 alkaline silicate 5 5 fluorescer 0.25 0.25 0.160.16 EDTA 0.15 0.15 0.18 0.18 SCMC 0.5 0.5 0.55 0.55 salt 2 2 sodiumsulphate 26.8 26.8 22.31 22.31 sodium carbonate 0 0 10.3 10.3 moisture10 10 11 11 TAED 3 3 3.3 3.3 sodium perborate 10 10 8 8 monohydratecalcium Dequest ® 2047 0.7 0.7 0.3 0.3 foam depressor 3 3 2.5 2.5perfume 0.2 0.2 0 0 alkaline protease 0.4 0.4 0.4 0.4 (Savinase (A) 6T)

Detergent composition Ingredients Ex. 1 Ex. 2 Ex. 3 Ex. 4 Level LevelLevel Level (parts (parts (parts (parts Material as is) as is) as is) asis) Glycerol 3.17 3.17 3.17 3.17 MPG 5.7 5.7 5.7 5.7 NaOH 2.13 2.13 2.132.13 TEA 2.05 2.05 2.05 2.05 Neodol 25-7 12.74 12.74 12.74 12.74 F-Dye0.18 0.18 0.18 0.18 Citric Acid 1.71 1.71 1.71 1.71 LAS (as LAS 8.498.49 8.49 8.49 Acid) Fatty acid 3.03 3.03 3.03 3.03 Empigen BB 1.5 1.51.5 1.5 SLES 4.24 4.24 4.24 4.24 Dequest 2066 0.875 0.875 0.875 0.875Patent Blue 0.00036 0.00036 0.00036 0.00036 Acid Yellow 0.00005 0.000050.00005 0.00005 Opacifier 0.0512 0.0512 0.0512 0.0512 Perfume 0.7340.734 0.734 0.734 Borax 10 10 10 10 Savinase 2.362 2.362 2.362 2.362Stainzyme 0.945 0.945 0.945 0.945 Soap 3.03 3.03 3.03 3.03 EPEI 20E0 (ex5.5 5.5 5.5 9 Nippon Shokubai) polyethyleneimine having a weight averagemolecular weight of about 600, and wherein the poly- ethyleneimine hasbeen modified by alkoxylation with an average 20 ethylene oxide moietiesLipex ® (ex 3 3 3 3 Novozymes) Texcare SRN170 0 7.5 0 0 (ex Clariant)soil release polymer Sokolan CPS 0 0 20 0 (ex BASF) Soil-release polymer

7.6. Methods of Assessing Amylase Activity in Detergent Compositions

Numerous α-amylase cleaning assays are known in the art, includingswatch and micro-swatch assays. The appended Examples describe only afew such assays.

In order to further illustrate the compositions and methods, andadvantages thereof, the following specific examples are given with theunderstanding that they are illustrative rather than limiting.

8. Brewing Compositions

The present variant amylase may be a component of a brewing compositionused in a process of brewing, i.e., making a fermented malt beverage.Non-fermentable carbohydrates form the majority of the dissolved solidsin the final beer. This residue remains because of the inability of maltamylases to hydrolyze the alpha-1,6-linkages of the starch. Thenon-fermentable carbohydrates contribute about 50 calories per 12 ouncesof beer. an amylase, in combination with a glucoamylase and optionally apullulanase and/or isoamylase, assist in converting the starch intodextrins and fermentable sugars, lowering the residual non-fermentablecarbohydrates in the final beer.

The principal raw materials used in making these beverages are water,hops and malt. In addition, adjuncts such as common corn grits, refinedcorn grits, brewer's milled yeast, rice, sorghum, refined corn starch,barley, barley starch, dehusked barley, wheat, wheat starch, torrifiedcereal, cereal flakes, rye, oats, potato, tapioca, and syrups, such ascorn syrup, sugar cane syrup, inverted sugar syrup, barley and/or wheatsyrups, and the like may be used as a source of starch.

For a number of reasons, the malt, which is produced principally fromselected varieties of barley, has the greatest effect on the overallcharacter and quality of the beer. First, the malt is the primaryflavoring agent in beer. Second, the malt provides the major portion ofthe fermentable sugar. Third, the malt provides the proteins, which willcontribute to the body and foam character of the beer. Fourth, the maltprovides the necessary enzymatic activity during mashing. Hops alsocontribute significantly to beer quality, including flavoring. Inparticular, hops (or hops constituents) add desirable bitteringsubstances to the beer. In addition, the hops act as proteinprecipitants, establish preservative agents and aid in foam formationand stabilization.

Grains, such as barley, oats, wheat, as well as plant components, suchas corn, hops, and rice, also are used for brewing, both in industry andfor home brewing. The components used in brewing may be unmalted or maybe malted, i.e., partially germinated, resulting in an increase in thelevels of enzymes, including α-amylase. For successful brewing, adequatelevels of α-amylase enzyme activity are necessary to ensure theappropriate levels of sugars for fermentation. an amylase, by itself orin combination with another α-amylase(s), accordingly may be added tothe components used for brewing.

As used herein, the term “stock” means grains and plant components thatare crushed or broken. For example, barley used in beer production is agrain that has been coarsely ground or crushed to yield a consistencyappropriate for producing a mash for fermentation. As used herein, theterm “stock” includes any of the aforementioned types of plants andgrains in crushed or coarsely ground forms. The methods described hereinmay be used to determine α-amylase activity levels in both flours andstock.

Processes for making beer are well known in the art. See, e.g., WolfgangKunze (2004) “Technology Brewing and Malting,” Research and TeachingInstitute of Brewing, Berlin (VLB), 3rd edition. Briefly, the processinvolves: (a) preparing a mash, (b) filtering the mash to prepare awort, and (c) fermenting the wort to obtain a fermented beverage, suchas beer. Typically, milled or crushed malt is mixed with water and heldfor a period of time under controlled temperatures to permit the enzymespresent in the malt to convert the starch present in the malt intofermentable sugars. The mash is then transferred to a mash filter wherethe liquid is separated from the grain residue. This sweet liquid iscalled “wort,” and the left over grain residue is called “spent grain.”The mash is typically subjected to an extraction, which involves addingwater to the mash in order to recover the residual soluble extract fromthe spent grain. The wort is then boiled vigorously to sterilizes thewort and help develop the color, flavor and odor. Hops are added at somepoint during the boiling. The wort is cooled and transferred to afermentor.

The wort is then contacted in a fermentor with yeast. The fermentor maybe chilled to stop fermentation. The yeast flocculates and is removed.Finally, the beer is cooled and stored for a period of time, duringwhich the beer clarifies and its flavor develops, and any material thatmight impair the appearance, flavor and shelf life of the beer settlesout. The beer usually contains from about 2% to about 10% v/v alcohol,although beer with a higher alcohol content, e.g., 18% v/v, may beobtained. Prior to packaging, the beer is carbonated and, optionally,filtered and pasteurized.

The brewing composition comprising an amylase, in combination with aglucoamylase and optionally a pullulanase and/or isoamylase, may beadded to the mash of step (a) above, i.e., during the preparation of themash. Alternatively, or in addition, the brewing composition may beadded to the mash of step (b) above, i.e., during the filtration of themash. Alternatively, or in addition, the brewing composition may beadded to the wort of step (c) above, i.e., during the fermenting of thewort.

A fermented beverage, such as a beer, can be produced by one of themethods above. The fermented beverage can be a beer, such as full maltedbeer, beer brewed under the “Reinheitsgebot,” ale, IPA, lager, bitter,Happoshu (second beer), third beer, dry beer, near beer, light beer, lowalcohol beer, low calorie beer, porter, bock beer, stout, malt liquor,non-alcoholic beer, non-alcoholic malt liquor and the like, but alsoalternative cereal and malt beverages such as fruit flavored maltbeverages, e.g., citrus flavored, such as lemon-, orange-, lime-, orberry-flavored malt beverages, liquor flavored malt beverages, e.g.,vodka-, rum-, or tequila-flavored malt liquor, or coffee flavored maltbeverages, such as caffeine-flavored malt liquor, and the like.

9. Reduction of Iodine-Positive Starch

Variant amylases may reduce the iodine-positive starch (IPS), when usedin a method of liquefaction and/or saccharification. One source of IPSis from amylose that escapes hydrolysis and/or from retrograded starchpolymer. Starch retrogradation occurs spontaneously in a starch paste,or gel on ageing, because of the tendency of starch molecules to bind toone another followed by an increase in crystallinity. Solutions of lowconcentration become increasingly cloudy due to the progressiveassociation of starch molecules into larger articles. Spontaneousprecipitation takes place and the precipitated starch appears to bereverting to its original condition of cold-water insolubility. Pastesof higher concentration on cooling set to a gel, which on ageing becomessteadily firmer due to the increasing association of the starchmolecules. This arises because of the strong tendency for hydrogen bondformation between hydroxy groups on adjacent starch molecules. See J. A.Radley, ed., STARCH AND ITS DERIVATIVES 194-201 (Chapman and Hall,London (1968)).

The presence of IPS in saccharide liquor negatively affects finalproduct quality and represents a major issue with downstream processing.IPS plugs or slows filtration system, and fouls the carbon columns usedfor purification. When IPS reaches sufficiently high levels, it may leakthrough the carbon columns and decrease production efficiency.Additionally, it may results in hazy final product upon storage, whichis unacceptable for final product quality. The amount of IPS can bereduced by isolating the saccharification tank and blending the contentsback. IPS nevertheless will accumulate in carbon columns and filtersystems, among other things. The use of variant amylases is expected toimprove overall process performance by reducing the amount of IPS.

All references cited herein are herein incorporated by reference intheir entirety for all purposes. In order to further illustrate thecompositions and methods, and advantages thereof, the following specificexamples are given with the understanding that they are illustrativerather than limiting.

EXAMPLES Example 1: Assays

In the following examples, various assays were used as set forth belowfor ease in reading. Any deviations from the protocols provided beloware indicated in the relevant sections. In these experiments, aspectrophotometer was used to measure the absorbance of the productsformed after the completion of the reactions.

A. Performance Index

The performance index (PI) compares the performance or stability of thevariant (measured value) and the standard enzyme (theoretical value) atthe same protein concentration. In addition, the theoretical values canbe calculated, using the parameters of the Langmuir equation of thestandard enzyme. A performance index (PI) that is greater than 1 (PI>1)indicates improved performance by a variant as compared to the standard(e.g., wild-type Bacillus sp. 707 α-amylase, also called Amy707 orAmy#707), while a PI of 1 (PI=1) identifies a variant that performs thesame as the standard, and a PI that is less than 1 (PI<1) identifies avariant that performs worse than the standard.

B. Protein Content Assay

This assay is performed using filtered culture supernatant from culturesgrown in microtiter plates (MTPs) over 3 days at 37° C. with shaking at300 rpm and 80% humidity. A fresh 96-well V-bottom MTP containing 50 μlsupernatant per well is used for the High Performance LiquidChromatography (HPLC) protein determination method.

For the 24-site SEL libraries described in Example 3, supernatants werediluted three-fold into 10 mM potassium phosphate buffer pH 7.25containing 5% acetonitrile and 10% sodium chloride and 10 μl of eachdiluted sample was analazyed. An Agilent 1100 (Hewlet Packard) HPLCequipped with a Swift™ RP-all PN 68-1030-041 column (Teledyne Isco,Inc.) was used. For the full SEL libraries described in Example 4,supernatants were diluted nine-fold into 25 mM MOPS, 0.1 mM CaCl₂, pH7.15, 10% TFA, and 20 μL of each diluted sample was analyzed. An Agilent1200 (Hewlet Packard) HPLC equipped with a Poroshell 300SB-C8 (AgilentTechnologies) column was used.

In both cases, the solvent system consists of 0.1% trifluoroacetic acidin aqueous phase and 0.07% trifluoroacetic acid in acetonitrile.Absorbance is read at 222 nm and protein concentration of samples isdetermined based on a calibration curve (18 ppm-400 ppm) using purifiedwild-type Amy707 protein.

C. Ceralpha α-Amylase Activity Assay

The Ceralpha α-amylase assay is performed using the Ceralpha HR Kit(Megazyme, Wicklow, Ireland). The assay involves incubating culturesupernatant with a substrate mixture under defined conditions, and thereaction is terminated (and color developed) by the addition of Trizmabase solution. The substrate is a mixture of the defined oligosaccharide“nonreducing-end blocked p-nitrophenyl maltoheptaoside” (BPNPG7) andexcess levels of glucoamylase and β-glucosidase (which have no action onthe native substrate due to the presence of the “blocking group”). Onhydrolysis of the oligosaccharide by endoacting α-amylase, the excessquantities of α-glucosidase and glucoamylase present in the mixture giveinstantaneous and quantitative hydrolysis of the p-nitrophenylmaltosaccharide fragment to glucose and free p-nitrophenol. Theabsorbance at 405 nm is measured, and this relates directly to the levelof α-amylase in the sample analysed.

The equipment used for this set of assays includes a Biomek FX Robot(Beckman Coulter); a SpectraMAX MTP Reader (type 340-Molecular Devices)and iEMS incubator/shaker (Thermo Scientific). In this assay system, thereagent and solutions used are:

-   -   1) p-nitrophenyl maltoheptaoside (BPNPG7) substrate (Megazyme        Ceralpha HR kit);    -   2) 50 mM MOPS, 50 mM NaCl, 0.1 mM CaCl₂, 0.005% TWEEN® 80        buffer, pH 7.15 (for the 24-site SEL libraries described in        Example 3) or 50 mM MOPS, 0.005% TWEEN® 80 buffer, pH 7 (for the        full SEL libraries described in Example 4); and 3) 200 mM Boric        acid/NaOH buffer, pH 10.2 (STOP buffer).

A vial containing 54.5 mg BPNPG7 substrate is dissolved in 10 ml ofmilliQ water. The amylase samples (fermentation supernatant) are dilutedin MOPS buffer. The assay is performed by adding 25 μl of dilutedamylase solution into the wells of a MTP followed by the addition of 25μl 5.45 mg/ml BPNPG7 substrate solution. The solutions are mixed and theMTP is sealed with a plate seal and placed in an incubator/shaker(iEMS-Thermo Scientific) for 30 minutes at 25° C. and 900 rpm. Thereaction is terminated by adding 50 μl STOP buffer and the absorbance isread at wavelength 405 nm in an MTP-Reader. A non-enzyme control is usedto correct for background absorbance values.

D. CS-28 Rice Starch Microswatch Assay

The principle of this α-amylase assay is the liberation of an orange-dyedue to the hydrolysis of rice starch incorporated in the microswatch.The absorbance at 488 nm is measured and this relates to the level ofamylase activity in the sample analysed, at the desired conditions (pH,temperature, and buffer).

The equipment used for this set of assays includes a Biomek FX Robot(Beckman Coulter), a SpectraMAX MTP Reader (type 340-Molecular Devices)and iEMS incubator/shaker (Thermo Scientific). In this assay system thereagent and solutions used are:

-   -   1) CS-28 Microswatches (rice starch, colored);    -   2) 25 mM HEPES, 2 mM CaCl₂, 0.005% TWEEN 80 buffer, pH 8.0 (for        the 24-site SEL libraries described in Example 3) or 10 mM        HEPES, 2 mM CaCl₂, 0.005% TWEEN 80 buffer, pH 8.0, conductivity        1 mS/cm (for the full SEL libraries described in Example 4);    -   3) 25 mM CAPS, 2 mM CaCl₂, 0.005% TWEEN 80 buffer, pH 10.0; and    -   4) 10 mM NaCl, 0.1 mM CaCl₂, 0.005% TWEEN 80 (Dilution buffer).

CS-28 Microswatches of 5.5 mm circular diameter were delivered by theCenter for Testmaterials (CFT, Vlaardingen, The Netherlands). Twomicroswatches are placed in each well of a 96-well MTP. The amylasesamples (fermentation supernatant) are tested at appropriateconcentrations in several conditions, pre-diluted in 10 mM NaCl, 0.1 mMCaCl₂, 0.005% TWEEN®80 solution:

-   -   1) pH 8 (25 mM HEPES buffer) and 16° C.; final amylase conc. in        assay <0.3 μg/ml;    -   2) pH 8 (25 mM HEPES buffer) and 32° C.; final amylase conc. in        assay <0.05 μg/ml;    -   3) pH 8 (25 mM HEPES buffer) and 50° C.; final amylase conc. in        assay <0.005 μg/ml;    -   4) pH 10 (25 mM CAPS buffer) and 16° C.; final amylase conc. in        assay <0.75 μg/ml; and    -   5) pH 10 (25 mM CAPS buffer) and 50° C.; final amylase conc. in        assay <0.0075 μg/ml.

The incubator/shaker is set at the desired temperature, 16° C. (coldstorage chamber or refrigerator), 32° C. or 50° C. The culturesupernatant samples are diluted in dilution buffer to 20× the desiredfinal concentration. 190 μl of either HEPES or CAPS buffer is added toeach well of a microswatch-MTP and subsequently 10 μl of enzyme solutionis added to each well resulting in a total volume of 200 μl/well. TheMTP is sealed with a plate seal and placed in the iEMS incubator/shakerand incubated for 60 minutes at 1150 rpm at the desired temperature(16°, 32° or 50° C.). Following incubation under the appropriateconditions, 100 μl of solution from each well is transferred to a newMTP, and the absorbance at 488 nm is measured using aMTP-spectrophotometer. Controls containing two microswatches and bufferbut no enzyme are included for background subtraction.

To calculate wash performance, the obtained absorbance value iscorrected for the blank value (obtained after incubation ofmicroswatches in the absence of enzyme), and the resulting absorbance isa measure of hydrolytic activity. A performance index (PI) is calculatedfor each sample. For the PI calculation for the wash performanceindices, a curvefit is made based on the wild-type Amy707 enzyme (SEQ IDNO: 3), using the Langmuir equation. Using the protein concentration ofthe variants, the expected performance based on the curvefit iscalculated. The observed performance is divided by the calculatedperformance and this is then divided by the performance of the wild-typeAmy707 enzyme (SEQ ID NO: 3).

E. Thermostability Assay—Determination of Initial and ResidualActivities

The thermostability of the amylase variant in relation to a referenceamylase (wild-type Amy707, SEQ ID NO: 3) is determined by incubating theamylase samples under defined conditions in MOPS buffer, pH 7.15. Thetemperature of the incubation is selected such that approximately 70% ofthe initial reference amylase activity is lost. The initial and residualamylase activities are determined using the Ceralpha α-amylase methoddescribed in section C above.

The equipment used for this set of assays includes a Biomek FX Robot(Beckman Coulter); a SpectraMAX MTP Reader (type 340-Molecular Devices)and iEMS incubator/shaker (Thermo Scientific). In this assay system, thereagent solutions used are:

-   -   1) p-nitrophenyl maltoheptaoside (BPNPG7) substrate (Megazyme        Ceralpha HR kit);    -   2) 10 mM NaCl, 0.1 mM CaCl₂, 0.005% TWEEN® 80 buffer (Dilution        buffer);    -   3) 50 mM MOPS, 50 mM NaCl, 0.1 mM CaCl₂, 0.005% TWEEN®80 buffer,        pH 7.15;    -   4) 200 mM Boric acid/NaOH buffer, pH 10.2 (STOP buffer); and    -   5) Amylase culture supernatants, containing 50-150 μg/ml        protein.

A “master dilution” plate is prepared by diluting the culturesupernatant 20× in dilution buffer, followed by a 42× dilution step inMOPS buffer. From the master dilution 25 μl is used to determine theinitial amylase activity and 100₁11 is used for heat incubation. The 100μl sample is put in each well of a 96 well PCR plate (VWR 211-0297) thatis sealed with an aluminum seal and incubated at 69° C. for 30 minutesin a Tetrad PCR block (Biorad). To determine the initial (t_(oo)) andresidual (t₃₀) activity, a 25 μl sample is transferred into a MTP,containing 25 μl of BPNPG7 solution per well and incubated at 25° C. for30 minutes. The Ceralpha α-amylase assay is performed as described abovein Section C.

For each variant, the ratio of the residual and initial amylaseactivities is used to calculate thermostability as follows:Thermostability=[t⁻³⁰ value]/[t⁻⁰⁰ value], so the thermostabilityactivity ratio is calculated based on enzyme activity after the heatincubation, divided by enzyme activity before the heat incubation. Theperformance index for thermostability is determined by dividing theactivity ratio of the variant enzyme, with that of the similarly treatedwild-type Amy707 enzyme (SEQ ID NO: 3). Thermostability assays were onlyperformed for the 24-site SEL libraries described in Example 3.

F. Detergent Stability Assay

The stability of the reference amylase and variants thereof is measuredafter incubation under defined conditions in the presence of 10%commercially purchased Persil Color detergent, Henkel (purchased in2008). The detergent is heat inactivated before use, and the initial andresidual amylase activities are determined using the Ceralpha α□-amylaseassay as described in section C above.

The equipment used for this set of assays includes a Biomek FX Robot(Beckman Coulter); a SpectraMAX MTP Reader (type 340-Molecular Devices)and iEMS incubator/shaker (Thermo Scientific). In this assay system, thereagent solutions used are:

-   -   1) p-nitrophenyl maltoheptaoside (BPNPG7) substrate (Megazyme        Ceralpha HR kit):    -   2) liquid detergent (HDL commercial product, enzyme—inactivated,        2 hrs at 60° C.);    -   3) 10.5% detergent in 25 mM HEPES buffer, pH 8.0;    -   4) 50 mM MOPS, 50 mM NaCl, 0.1 mM CaCl₂, 0.005% TWEEN®80 buffer,        pH 7.15 (for the 24-site SEL libraries described in Example 3)        or 50 mM MOPS, 0.1 mM CaCl₂, 0.005% TWEEN®80 buffer, pH 7 (for        the full SEL libraries described in Example 4);    -   5) 200 mM Boric acid/NaOH buffer, pH 10.2 (STOP buffer); and    -   6) Amylase culture supernatants containing 50-150 μg/ml protein.

To a 96 well PCR plate, 95 μl of a 10.5% detergent solution is added,and mixed with 5 μl of culture supernatant. A 3 μl aliquot is removedfor determination of the initial amylase activity. The PCR plate isincubated in on a Tetrad PCR block at 41° for 30 minutes. Afterincubation the residual amylase activity is measured using 3 μl of thedetergent-enzyme mixture. To determine the initial (t₀) and residual(t₃₀) amylase activity, 3 μl ‘detergent-enzyme’ mix is diluted in 122 μlMOPS buffer and subsequently 25 μl is used to determine the amylaseactivity using the Ceralpha α-amylase assay described above in sectionC.

For each variant, the ratio of the residual and initial amylaseactivities is used to calculate the detergent stability as follows:Detergent stability=[t⁻³⁰ value]/[t⁻⁰⁰ value], so the detergentstability activity ratio is calculated based on enzyme activity afterthe heat incubation, divided by enzyme activity before the heatincubation.

For each sample (variants) the performance index (PI) is calculated. Theperformance index for detergent stability is determined by comparing thedetergent stability of the variant enzyme, with that of the similarlytreated wild-type Amy707 enzyme (SEQ ID NO: 3).

Example 2: Generation of B. subtilis Strains Expressing Amy707 andVariants Thereof

In this example, the construction of Bacillus subtilis strainsexpressing wild-type Amy707 α-amylase and variants, thereof, aredescribed. Amy707 is the G6-amylase (1,4-α-D-glucan maltohexaohydrolase)of alkalophilic Bacillus sp. #707 for which the nucleotide sequence wasdescribed by Tsukamoto et al. (1988) Biochem. Biophys. Res. Commun. 151:25-31.

A synthetic DNA fragment (SEQ ID NO: 1, herein referred to as “Amy707DNA”) encoding Amy707 (SEQ ID NO: 3) α-amylase was produced by GENEARTAG (Regensburg, Germany) and served as template DNA for the constructionof Bacillus subtilis strains expressing Amy707 α-amylase and variants,thereof.

SEQ ID NO:1 includes a codon-modified nucleotide sequence encoding themature form of Amy707 α-amylase adjacent to a sequence encoding the LATsignal peptide (underlined):

ATGAAACAACAAAAACGGCTTTACGCCCGATTGCTGACGCTGTTATTTGCGCTCATCTTCTTGCTGCCTCATTCTGCAGCTTCAGCACATCATAATGGCACAAACGGCACGATGATGCAGTATTTTGAATGGTATCTGCCGAACGATGGAAACCATTGGAACCGCCTGAATAGCGATGCGAGCAACCTGAAAAGCAAAGGCATCACAGCAGTTTGGATTCCGCCGGCATGGAAAGGAGCAAGCCAAAACGACGTCGGCTATGGAGCGTATGATCTGTATGACCTGGGCGAATTTAACCAAAAAGGCACGGTCCGCACGAAATATGGCACGCGCAGCCAACTTCAAGCAGCAGTCACGAGCCTTAAAAACAACGGCATCCAGGTCTATGGAGATGTCGTCATGAACCATAAAGGCGGAGCAGATGCGACAGAAATGGTCAGAGCGGTCGAAGTCAACCCGAACAACCGCAATCAAGAAGTCACGGGCGAATATACAATCGAAGCGTGGACGCGCTTTGATTTTCCGGGCAGAGGCAATACACATAGCAGCTTTAAATGGCGCTGGTATCATTTTGATGGCGTCGATTGGGATCAAAGCCGCAGACTGAACAACCGCATCTATAAATTTCGCGGCCATGGCAAAGCATGGGATTGGGAAGTCGATACGGAAAACGGCAACTATGACTATCTGATGTATGCGGACATCGATATGGATCATCCGGAAGTCGTCAACGAACTGAGAAATTGGGGCGTCTGGTATACAAATACGCTGGGCCTGGATGGCTTTAGAATCGACGCGGTCAAACATATCAAATATAGCTTTACGCGCGACTGGATCAATCATGTCAGAAGCGCGACGGGCAAAAATATGTTTGCGGTCGCGGAATTTTGGAAAAATGATCTGGGCGCGATCGAAAACTATCTGCAAAAAACGAACTGGAACCATAGCGTCTTTGATGTCCCGCTGCATTATAACCTGTATAACGCGAGCAAAAGCGGCGGCAATTATGATATGCGCAACATCTTTAACGGCACGGTCGTTCAAAGACATCCGAGCCATGCGGTCACGTTTGTCGATAACCATGATAGCCAACCGGAAGAAGCGCTGGAAAGCTTTGTCGAAGAATGGTTTAAACCGCTGGCGTATGCACTGACACTGACGAGAGAACAAGGATATCCGAGCGTCTTTTATGGCGACTATTATGGCATCCCGACACATGGAGTTCCGGCGATGAGAAGCAAAATCGACCCGATCCTGGAAGCGAGACAGAAATATGCGTATGGCAAACAGAACGACTATCTGGACCATCATAACATCATCGGCTGGACGAGAGAAGGAAATACGGCGCATCCGAATTCAGGACTGGCGACGATTATGTCAGATGGAGCGGGCGGAAGCAAATGGATGTTTGTCGGCAGAAACAAAGCAGGACAAGTCTGGAGCGATATCACGGGCAATAGAACGGGAACGGTCACGATCAATGCAGATGGCTGGGGCAACTTTAGCGTTAATGGCGGAAGCGTCAGCATCTGGGTCAACAAA

The precursor form of the Amy707 polypeptide produced from thepHPLT-Amy707 vector is shown, below, as SEQ ID NO: 2. The LAT signalpeptide is underlined:

MKQQKRLYARLLTLLFALIFLLPHSAASAHHNGTNGTMMQYFEWYLPNDGNHWNRLNSDASNLKSKGITAVWIPPAWKGASQNDVGYGAYDLYDLGEFNQKGTVRTKYGTRSQLQAAVTSLKNNGIQVYGDVVMNHKGGADATEMVRAVEVNPNNRNQEVTGEYTIEAWTRFDFPGRGNTHSSFKWRWYHFDGVDWDQSRRLNNRIYKFRGHGKAWDWEVDTENGNYDYLMYADIDMDHPEVVNELRNWGVWYTNTLGLDGFRIDAVKHIKYSFTRDWINHVRSATGKNMFAVAEFWKNDLGAIENYLQKTNWNHSVFDVPLHYNLYNASKSGGNYDMRNIFNGTVVQRHPSHAVTFVDNHDSQPEEALESFVEEWFKPLAYALTLTREQGYPSVFYGDYYGIPTHGVPAMRSKIDPILEARQKYAYGKQNDYLDHHNIIGWTREGNTAHPNSGLATIMSDGAGGSKWMFVGRNKAGQVWSDITGNRTGTVTINADGWGN FSVNGGSVSIWVNK

The mature form of the Amy707 polypeptide produced from the pHPLT-Amy707vector is shown, below, as SEQ ID NO: 3.

HHNGTNGTMMQYFEWYLPNDGNHWNRLNSDASNLKSKGITAVWIPPAWKGASQNDVGYGAYDLYDLGEFNQKGTVRTKYGTRSQLQAAVTSLKNNGIQVYGDVVMNHKGGADATEMVRAVEVNPNNRNQEVTGEYTIEAWTRFDFPGRGNTHSSFKWRWYHFDGVDWDQSRRLNNRIYKFRGHGKAWDWEVDTENGNYDYLMYADIDMDHPEVVNELRNWGVWYTNTLGLDGFRIDAVKHIKYSFTRDWINHVRSATGKNMFAVAEFWKNDLGAIENYLQKTNWNHSVFDVPLHYNLYNASKSGGNYDMRNIFNGTVVQRHPSHAVTFVDNHDSQPEEALESFVEEWFKPLAYALTLTREQGYPSVFYGDYYGIPTHGVPAMRSKIDPILEARQKYAYGKQNDYLDHHNIIGWTREGNTAHPNSGLATIMSDGAGGSKWMFVGRNKAGQVWSDITGNRTGTVTINADGWGNFSVNGGSVSIWVNK

To express Amy707, the Amy707 DNA fragment was cloned into the pHPLTvector (Solingen et al. (2001) Extremophiles 5:333-341) by GENEART andfused in-frame to the AmyL (LAT) signal peptide using the unique PstIand HpaI restriction sites, resulting in plasmid pHPLT-Amy707. The pHPLTexpression vector contains the B. licheniformis LAT promoter (Plat) andadditional elements from pUB110 (McKenzie et al. (1986) Plasmid, 15:93-103) including a replicase gene (reppUB), a neomycin/kanamycinresistance gene (neo) and a bleomycin resistance marker (bleo). A map ofthe pHPLT vector containing the Amy707 gene (pHPLT-Amy707) is shown inFIG. 2.

A suitable B. subtilis strain was transformed with pHPLT-Amy707 plasmidDNA using a method known in the art (WO 02/14490). The B. subtilistransformants were selected on agar plates containing heart infusionagar (Difco, Catalog No. 244400) and 10 mg/L neomycin sulfate (Sigma,Catalog No. N-1876; contains 732 μg neomycin per mg). Selective growthof B. subtilis transformants harboring the pHPLT-Amy707 plasmid wasperformed in shake flasks containing MBD medium (a MOPS based definedmedium), 5 mM CaCl₂ and 10 mg/L neomycin. Growth resulted in theproduction of secreted Amy707 amylase with starch hydrolyzing activity.

Example 3: Generation and Evaluation of a 24-Site Amy707 Site EvaluationLibraries

A. Generation of the Library

Site evaluation libraries (SELs) were created by GENEART using aproprietary process (WO 2004/059556A3), and using methods and devicesfor optimizing a nucleotide sequence for the purpose of expression of aprotein by PCR, and the manufacture of DNA molecules utilized technologyowned by or licensed to GENEART (European Patent Nos. 0 200 362 and 0201 184; and U.S. Pat. Nos. 4,683,195, 4,683,202 and 6,472,184). Theconstruction of Amy707 SELs described in this example was performed byGENEART using their technology platform for gene optimization, genesynthesis and library generation under proprietary GENEART know howand/or intellectual property. The sequential permutation approach ofGENEART, to produce SELs, is described in general on the company's website.

The pHPLT-Amy707 plasmid DNA served as template to produce SELs atpre-selected sites in the mature region (SEQ ID NO: 3) shown on Table3.1. GENEART was commissioned to create the SELs at those positionsusing their standard protocols. The corresponding codons for each sitewere each substituted with codons for at least 16 (out of a possible 19)different amino acids. The codon-mutagenized pHPLT-Amy707 mixes wereused to transform competent B. subtilis cells as known in the art (WO2002/014490) to generate the Amy707 SELs. Transformation mixes wereplated on HI-agar plates (Heart Infusion agar) containing 10 mg/Lneomycin sulfate. For each library, single bacterial colonies werepicked and grown in TSB (tryptone and soy-based broth) liquid mediumwith 10 mg/ml neomycin selection for subsequent DNA isolation and genesequence analysis. Sequence analysis data revealed a maximum of 19Amy707 mature variants per library. To generate Amy707 and variantenzyme samples for biochemical characterization, selective growth of thevariants was performed in 96 well MTPs at 37° C. for 68 hours in MBDmedium. A total of 402 out of the 456 possible variants were obtainedfor the 24 positions mutagenized.

TABLE 3.1 Amy707 Site Evaluation Library Positions. H001 S083 N125 N128V131 Y160 K179 H183 G184 A186 E190 S244 Q280 N306 R320 H321 P380 H408A434 I454 N475 G476 G477 N484B. Identification of Combinable and Productive Mutations

Performance index (PI) values were determined for all the Amy707 amylasevariants tested using the assays described in Example 1: α-amylaseactivity, CS-28 microswatch assay (at both pH8 and pH10), detergentstability, thermostability assays, and protein determination.

Productive positions are described as those positions within a moleculethat are most useful for making combinatorial variants exhibiting animproved characteristic, where the position itself allows for at leastone combinable mutation. Combinable mutations are mutations at any aminoacid position that can be used to make combinatorial variants.Combinable mutations improve at least one desired property of themolecule, while not significantly decreasing either expression,activity, or stability. Combinable mutations can be grouped as follows:

-   -   Group A: A mutation that produces a variant wherein the minimum        performance indices (PI) relative to a defined parental protein        for: (i) protein expression, (ii) activity, (iii) CS-28        microswatch activity at pH 8 (16° C., 32° C., or 50° C.) or pH10        (16° C. or 50° C.), and (iv) detergent stability or        thermostability are greater than or equal to 0.9, and in        addition have a PI for any one of these tests that is greater        than or equal to 1.0.    -   Group B: A mutation that produces a variant wherein the minimum        performance indices (PI) relative to a defined parental protein        for: (i) protein expression, (ii) activity, (iii) CS-28        microswatch activity at pH 8 (16° C., 32° C., or 50° C.) or pH10        (16° C. or 50° C.), and (iv) detergent stability or        thermostability are greater than or equal to 0.8, and in        addition have a PI for any one of these tests that is greater        than or equal to 1.2.    -   Group C: A mutation that produces a variant wherein the minimum        performance indices (PI) relative to a defined parental protein        for: (i) protein expression, (ii) activity, (iii) CS-28        microswatch activity at pH 8 (16° C., 32° C., or 50° C.) or pH10        (16° C. or 50° C.), and (iv) detergent stability or        thermostability are greater than or equal to 0.5, and in        addition have a PI for any one of these tests that is greater        than or equal to 1.5.

The properties of combinable mutations are summarized in the followingTable.

TABLE 3.2 Properties for each group of combinable mutations PerformanceIndex (PI) Synthetic Stability Minimum PI Cleaning substrate (detergentor in one or Group Expression (pH 6 or 8) activity thermal) more tests A≥0.9 ≥0.9 ≥0.9 ≥0.9 X ≥ 1.0 B ≥0.8 ≥0.8 ≥0.8 ≥0.8 X ≥ 1.2 C ≥0.5 ≥0.5≥0.5 ≥0.5 X ≥ 1.5

Preferred combinable mutations are at “productive positions,” asdescribed, below. In the case of the present α-amylases, “activity”refers to α-amylase activity, which can be measured as described,herein.

Productive positions are amino acid positions that are tolerant tosubstitution with different amino acid residues, wherein the resultingvariants meet a set of performance criteria for combinability, as setforth above. Productive positions can be assigned a Productivity Scoreas follows: Positions where less than 15% of the substitutions at agiven position fall within groups A, B, or C are given a ProductivityScore of “1”. Positions where less than 40%, but greater than, or equalto 15% of the substitutions at a given position fall within groups A, B,or C are given a Productivity Score of “2”. Positions where less than75%, but greater than, or equal to 40% of the substitutions at a givenposition fall within groups A, B, or C are given a Productivity Score of“3”. Positions where 75% or more of the substitutions at a givenposition fall within groups A, B, or C are given a Productivity Score of“4”. Preferred productive positions are combinable mutations.

Suitability score refers to the ability of one or more combinablemutations to be used to make combinatorial variants, based on theperformance criteria for combinability, (i.e., A, B, and C, as setforth, above) in which each of the mutations fall. A higher suitabilityscore indicates a mutation or mutations that are more suitable for usein making combinatorial variants. Suitability scores are described inthe following Table.

TABLE 3.3 Definitions of suitability scores Substitutions Occur inGroup(s) Suitability Score A, B and C +++++ A and B ++++ A or (B and C)+++ B ++ C +

Table 3.4 shows the Productivity Score (4, 3, or 2,) calculated for eachposition in the Amy707 protein. No positions were calculated to have aproductivity score of 1. For each Amy707 position, variants are listedaccording to the suitability score they received (+, ++, +++, ++++, or+++++). Position numbering is based on the mature Amy707 protein listedin SEQ ID NO: 3.

TABLE 3.4 Productivity Score for each position in the Amy707 protein.Productivity VARIANTS SUITABILITY SCORE POS score (+) (++)  (+++)*(++++) (+++++) 1 3 HILTFWQ ACK RM 83 3 A SCTNG IRK 125 4 RTY NLMVGH ISFWC 128 2 EY D NL C 131 2 RKS VCT 160 4 L YIG ARKSHQ CDEN 179 3 LEVNW KICMQ G 183 4 T HGLF RCSDEMVNW AP YQ 184 3 I ALQ G CN DE 186 2 R ADSG EMN244 2 STN KDEHQ 280 2 CT QDE IKN 306 3 NIKDTEVG AR 320 3 HQ RDEN AST K321 2 MV HFY 380 3 PDTEGHQ C KS 408 3 HIMNPQ RSTEG K 434 4 L AIRDEMGPHQCKSTVN 454 2 M ICSV 475 2 NRCSD 476 2 GRNHQ CDE 477 2 T A GKDNQ R 484 3W DG NTQ ARS *The first listed amino acid residue is the wild-typeresidue.

Example 4: Generation and Evaluation of a Full Amy707 Site EvaluationLibraries

A. Generation of the Libraries

Site evaluation libraries (SELs) were created by GENEART using aproprietary process (WO 2004/059556A3), and using methods and devicesfor optimizing a nucleotide sequence for the purpose of expression of aprotein by PCR, and the manufacture of DNA molecules utilized technologyowned by or licensed to GENEART (European Patent Nos. 0 200 362 and 0201 184; and U.S. Pat. Nos. 4,683,195, 4,683,202 and 6,472,184). Theconstruction of Amy707 SELs described in this example was performed byGENEART using their technology platform for gene optimization, genesynthesis and library generation under proprietary GENEART know howand/or intellectual property. The sequential permutation approach ofGENEART, to produce SELs, is described in general on the company's website.

The pHPLT-Amy707 plasmid DNA served as template to produce SELs at allsites in the mature region (SEQ ID NO: 3). GENEART was commissioned tocreate the SELs at these positions using their standard protocols. Thecorresponding codons for each site were each substituted with codons forat least 16 (out of a possible 19) different amino acids. Thecodon-mutagenized pHPLT-Amy707 mixes were used to transform competent B.subtilis cells as known in the art (WO 2002/014490) to generate theAmy707 SELs. Transformation mixes were plated on HI-agar plates (HeartInfusion agar) containing 10 mg/L neomycin sulfate. For each library,single bacterial colonies were picked and grown in TSB (tryptone andsoy-based broth) liquid medium with 10 mg/ml neomycin selection forsubsequent DNA isolation and gene sequence analysis. Sequence analysisdata revealed a maximum of 19 Amy707 mature variants per library. Togenerate Amy707 and variant enzyme samples for biochemicalcharacterization, selective growth of the variants was performed in 96well MTPs at 37° C. for 68 hours in MBD medium.

B. Identification of Combinable and Productive Mutations

Performance index (PI) values were determined for all the Amy707 amylasevariants tested using the assays described in Example 1: α-amylaseactivity, CS-28 microswatch assay (at both pH8 and pH10), detergentstability, thermostability assays, and protein determination.

Productive positions are described as those positions within a moleculethat are most useful for making combinatorial variants exhibiting animproved characteristic, where the position itself allows for at leastone combinable mutation. Combinable mutations are mutations at any aminoacid position that can be used to make combinatorial variants.Combinable mutations improve at least one desired property of themolecule, while not significantly decreasing either expression,activity, or stability. Combinable mutations can be grouped as follows:

-   -   Group A: A mutation that produces a variant wherein the minimum        performance indices (PI) relative to a defined parental protein        for: (i) protein expression, (ii) activity, (iii) CS-28        microswatch activity at pH 8 (16° C., 32° C., or 50° C.) or pH10        (16° C. or 50° C.), and (iv) detergent stability are greater        than or equal to 0.9, and in addition have a PI for any one of        these tests that is greater than or equal to 1.0.    -   Group B: A mutation that produces a variant wherein the minimum        performance indices (PI) relative to a defined parental protein        for: (i) protein expression, (ii) activity, (iii) CS-28        microswatch activity at pH 8 (16° C., 32° C., or 50° C.) or pH10        (16° C. or 50° C.), and (iv) detergent stability are greater        than or equal to 0.8, and in addition have a PI for any one of        these tests that is greater than or equal to 1.2.    -   Group C: A mutation that produces a variant wherein the minimum        performance indices (PI) relative to a defined parental protein        for: (i) protein expression, (ii) activity, (iii) CS-28        microswatch activity at pH 8 (16° C., 32° C., or 50° C.) or pH10        (16° C. or 50° C.), and (iv) detergent stability are greater        than or equal to 0.5, and in in addition have a PI for any one        of these tests that is greater than or equal to 1.5.

The properties of combinable mutations are summarized in the followingTable.

TABLE 4.1 Properties for each group of combinable mutations PerformanceIndex (PI) Synthetic Minimum PI Cleaning substrate Detergent in one orGroup Expression (pH 8 or 10) activity Stability more tests A ≥0.9 ≥0.9≥0.9 ≥0.9 X ≥ 1.0 B ≥0.8 ≥0.8 ≥0.8 ≥0.8 X ≥ 1.2 C ≥0.5 ≥0.5 ≥0.5 ≥0.5 X≥ 1.5

Preferred combinable mutations are at “productive positions,” asdescribed, below. In the case of the present α-amylases, “activity”refers to α-amylase activity, which can be measured as described,herein.

Productive positions are amino acid positions that are tolerant tosubstitution with different amino acid residues, wherein the resultingvariants meet a set of performance criteria for combinability, as setforth above. Productive positions can be assigned a Productivity Scoreas follows: Positions where less than 15% of the substitutions at agiven position fall within groups A, B, or C are given a ProductivityScore of “1”. Positions where less than 30%, but greater than, or equalto 15% of the substitutions at a given position fall within groups A, B,or C are given a Productivity Score of “2”. Positions where less than50%, but greater than, or equal to 30% of the substitutions at a givenposition fall within groups A, B, or C are given a Productivity Score of“3”. Positions where 50% or more of the substitutions at a givenposition fall within groups A, B, or C are given a Productivity Score of“4”. Preferred productive positions are combinable mutations.

Suitability score refers to the ability of one or more combinablemutations to be used to make combinatorial variants, based on theperformance criteria for combinability, (i.e., A, B, and C, as setforth, above) in which each of the mutations fall. A higher suitabilityscore indicates a mutation or mutations that are more suitable for usein making combinatorial variants. Suitability scores are described inthe following Table.

TABLE 4.2 Definitions of suitability scores Substitutions OccurSuitability in Group(s) Score A, B and C +++++ A and B ++++ A or (B andC) +++ B ++ C +

Table 4.3 shows the Productivity Score (Prod. Score, 4, 3, 2, or 1)calculated for each position in the Amy707 protein. For each Amy707position, variants are listed according to the suitability score theyreceived (+, ++, +++, ++++, or +++++). Position numbering is based onthe mature Amy707 protein listed in SEQ ID NO: 3.

TABLE 4.3 Productivity Score for each position in the Amy707 protein.Prod. VARIANTS SUITABILITY SCORE POS Score (+) (++) (+++)* (++++)(+++++) 1 4 E C HFKNQRT AILMW 2 4 A HCDEFGIKN LM PQSW 3 4 NCDFKLQSTV AEM4 4 DKM GFHPSTW EIL 5 4 CN THIQSVW DGM A 6 3 EGQT NS A 7 4 A DILTY GMSHPQRV 10 1 IL M 12 1 Y A 16 3 HT YAENW D 17 3 D V LS AGT 18 1 AE P 19 1L N D 20 3 EHI DS GNY AC 22 4 GMV I NELQSTW R 23 2 HQ FMT 25 4 M NS TVACGKY 26 2 RKQT 27 2 A LIV 28 4 NADQWY CEGHKR 29 4 SCDEFHKM AN RTVWY 302 DEMNQR 31 1 AS 32 4 CDEG SN MWY ILQR 33 4 NHIKQTVWY CDMR 34 1 LFM 35 4CEFIL KMNQ AGH 36 2 SDGKQT 40 1 K T N 41 3 IKM DQ A S C 47 2 GMP AS 50 2GC S 52 2 L ST KM R 53 1 Q A 54 4 M NADEFGQS C VW 56 2 NS V E 61 1 Y F63 2 NQ LM 64 1 YH 66 1 V L M 68 2 D EA Q 70 4 R NEGHKV CDFIMS L 72 1 KR 73 4 D EW GQRT KMSY 74 2 G T S 75 1 M VI 77 2 A S TI NV 81 3TACDFIKNPS 82 3 Q RACFSVY IKM 83 2 M SKNQRT 84 3 CFL QEN DK M 86 4 HQEIRTVWY K 87 3 ADKT M 88 1 A M 89 2 VAC I 90 2 TGMQRS 91 3 M SHKQRTVAEN 93 1 H KR 94 4 NFH ACDGKLM QR 95 4 G D NCFHIQRSTY A 96 2 DEN G 97 1V I 98 3 QCDEGHKR A 99 2 A VC I 100 2 Y CFI 101 1 A G 103 2 F I VL CT A110 2 PS GA 111 1 S A 112 1 C D E 113 4 IVY A CEFGHKMR 115 1 E Q 116 4PV MDIQ ACEFGL T NRW 117 3 L VEPS R T 118 3 DLV RW EQT G 119 1 AC S 1221 VC 123 2 ACL N 124 1 NT P 125 4 Y NGHISTW CFLMR 126 1 N D 128 2 E N LYC 129 1 V Q 132 1 TS 133 3 GAHQST DP 134 2 V ES DT P 135 2 Q YFL CM 1364 FY TCKLQR DGMNP 138 2 DLMN E 139 2 CG A 140 1 WF Y 142 3 T RS CEFGHKY144 3 DEISY KM 145 1 FMY 146 4 ACDEF P GHMR SWY 147 2 GDIL A 149 4 PWGCDEFHKRV AL 150 2 L HM NPS R 151 4 D TEGHILMQV 153 1 N S 154 2 LRY S155 1 W F 156 2 AD KS 158 2 AL RKQ CN 160 4 GILMP F YAC HQS DEKNR 162 1M F 165 1 V CT 167 1 M F W 168 1 C D 169 4 FGIKWY S QCMN ADEHV 170 2 SCAEK 171 2 RT MS 172 4 Y RCEGHQ AMS 173 4 DKN L ACFHWY 174 4 NDGHILPSTV175 3 M NRS ADEL 176 1 KT R 178 1 W Y 179 2 CL KM Q 181 4 ALNT REF CIMQVSY 182 1 C GD 183 4 N HCQVWY DEFLMP A 184 2 AEL D G N C 186 2 AG EN D195 2 FY NW L 196 1 G C 203 1 Y N 206 2 NS H IM C T 210 3 ACEM H D NQRS211 4 L P CDMNQS AFKV 215 3 FL NKM DQ 216 3 GLN EY ACST HQ 217 1 M L 2183 ACEFHM K R 219 3 ACM NRT D 221 1 IV G 222 4 EM VDFGNY AHILRST 225 2 ATR K 226 2 Y NDE AK 227 2 TE AK 228 1 L IM 229 2 FV C G 230 1 M L 231 2DE GT 233 3 L CHMY FAW 235 2 ILMV 238 1 I V 243 1 YF 244 2 S N DEHQ 2451 E M F 247 2 L RT AE 249 2 M FL W 250 2 ILMV 251 4 AH NPTVY CGW KLQRS252 2 HDEKN 253 1 M V 257 2 TMS A 258 3 IMV PS GDN K R 259 3 DERT KCPAGHQ 260 2 N DP KR 261 3 G MCI AEQT 262 4 ACDEG HKRY FM LQS 263 1 AS 2651 AS G 273 4 CK GV DEHLMPQS TY 276 1 C ET 280 2 MV QDHK N 283 1 NG D 2852 NEKST M 286 3 F N HLV ACEM T 287 2 ADNY S 288 2 L C V IT 292 1 LM P296 1 NQ 297 1 L M 298 2 YFRW 299 3 MY NGHRST 301 1 SAG 302 2 T KCM EQSR 303 4 SEQR ACDGLM 304 2 C GKRSV 306 2 NG AD 307 1 YAF 310 1 RQS 311 4NFT DEGHKLM QRY 312 2 L IV M 313 1 MY F 314 4 M NADEGHIKL Q STVY 317 1 LV 318 2 MS VCI LT 319 3 Y QADEGHNR 320 4 DY RHNST AEKMQ 321 1 HWY 322 1D P 323 4 A P SFHIL CGRVY DEMT 324 2 ACM K H Y 326 3 H C VNT AM 327 1 TL 328 1 FV 329 1 V I 334 1 T S 337 3 Y A ECDST NQ 339 2 GT A S 341 2 FGKEH ADY 343 1 FTY 344 1 VI C 345 4 EAGHKLMN QSTY 346 4 E ACHKRTVY DGMNQS347 1 A D W 350 1 PE 351 2 AC LM Q 352 1 AS 354 1 AS 355 2 LIKMV 356 2 QCV T IL 357 2 H A L M 358 2 AI G T C 359 2 I W RV 360 4 EACFHLNPQ K RTVY361 4 V HT QDSW C AEG 363 3 IW V YE ADKNQ M 364 2 CG P A 365 2 GV S AN366 2 C V IL 367 1 FY 368 2 GL Y MQ 369 1 S A G 372 3 R YHIKM QTV 374 2C IQ NS 375 4 Q PDGHIRTVY AEKM 377 2 A HKM GT 378 2 L GEHMN 379 3 ALVIQS MNRY 380 2 PDEGHKQS 381 2 AGNQRST 382 1 K M 386 1 LV I 387 2 EN DG388 4 HNS PACFGKLQR D TVY 389 3 F IV EGLMQS 390 1 M LV 391 4 S ECHIWAGKLNR 392 2 ACG S 394 3 EH QCDGLRV Y 395 4 V KDEMST AGQR 396 2 YN K M397 1 AG 400 4 AI KF GHLMQTVW 401 1 QH M 402 3 V NCIKLSY 403 1 ET D 4052 A LCMNTV 406 2 ACL Q D N 408 4 HEGNQRST KMP 410 1 N I 411 1 I V 412 1GAS 413 2 WFHIY L 414 2 TACV S 415 2 W CY R 416 4 EFHQRTVWY ADGKLN 417 1A G 418 4 I NADKMQSTV L 419 4 TDEHKLNP MSY QRW 420 4 DFGHI A LMQRS TVW421 4 V HCI DEKLMRWY AN 422 4 A PGVY CEFLMT 423 4 NCDEFHILR ST 424 4 NEV SCDG AIQTW 425 1 GA 426 2 L ANS 427 1 ACT 428 1 TNS 429 1 IM 430 2MGIL V 431 1 AC S 433 3 M C GDEKNR A 434 4 ACDEFHIKN M PQRSTV 435 4 ETGKMQR ACNP 436 2 ACQ D GS 437 3 C SKNT AD 438 2 CE KS H 439 2 L WH MQ441 2 FH NY 442 1 A VC 444 2 AQ RK 445 3 C NGKRT AEQ 446 3 T Y KCFS AHMQ448 1 F N G 450 3 AL VEIQRST 451 1 WF 452 4 H SCEFQTW AKNY 454 3 F CMSIAV L 457 2 T NGHQR 458 4 L D RHSTVY CEKMN 459 4 ADEGH CP TL NS 460 3 KNGQ EHS 461 4 F TDV ACEGKLNP QRY 463 2 L TEKPQR 465 2 Q N DG 466 4ADEGKNPQ RS 467 1 D E 469 1 Y W 471 3 NHQRY CDE 473 1 SP 474 1 V S 475 1NDE 476 2 HN GDR E 477 3 PT DK GAR NQ 478 1 A SG 479 1 VT 481 2 I LTV482 1 WY 483 3 VHMR CGST 484 3 NAEGHQRS 485 3 MP KHQST *The first listedamino acid residue is the wild-type residue.

The productive positions in Amy 707 that fall within the previouslydescribed Productivity Scores of “4” and the substitutions within thosepositions that are combinable are listed below. Position numbering isbased on the mature Amy707 protein listed in SEQ ID NO: 3.

1(H, A, C, E, F, I, K, L, M, N, Q, R, T, W); 2(H, A, C, D, E, F, G, I,K, L, M, N, P, Q, S, W); 3(N, A, C, D, E, F, K, L, M, Q, S, T, V); 4(G,D, E, F, H, I, K, L, M, P, S, T, W); 5(T, A, C, D, G, H, I, M, N, Q, S,V, W); 7(G, A, D, H, I, L, M, P, Q, R, S, T, V, Y); 22(N, E, G, I, L, M,Q, R, S, T, V, W); 25(N, A, C, G, K, M, S, T, V, Y); 28(N, A, C, D, E,G, H, K, Q, R, W, Y); 29(S, A, C, D, E, F, H, K, M, N, R, T, V, W, Y);32(S, C, D, E, G, I, L, M, N, Q, R, W, Y); 33(N, C, D, H, I, K, M, Q, R,T, V, W, Y); 35(K, A, C, E, F, G, H, I, L, M, N, Q); 54(N, A, C, D, E,F, G, M, Q, S, V, W); 70(N, C, D, E, F, G, H, I, K, L, M, R, S, V);73(G, D, E, K, M, Q, R, S, T, W, Y); 86(Q, E, H, I, K, R, T, V, W, Y);94(N, A, C, D, F, G, H, K, L, M, Q, R); 95(N, A, C, D, F, G, H, I, Q, R,S, T, Y); 113(A, C, E, F, G, H, I, K, M, R, V, Y); 116(M, A, C, D, E, F,G, I, L, N, P, Q, R, T, V, W); 125(N, C, F, G, H, I, L, M, R, S, T, W,Y); 136(T, C, D, F, G, K, L, M, N, P, Q, R, Y); 146(P, A, C, D, E, F, G,H, M, R, S, W, Y); 149(G, A, C, D, E, F, H, K, L, P, R, V, W); 151(T, D,E, G, H, I, L, M, Q, V); 160(Y, A, C, D, E, F, G, H, I, K, L, M, N, P,Q, R, S); 169(Q, A, C, D, E, F, G, H, I, K, M, N, S, V, W, Y); 172(R, A,C, E, G, H, M, Q, S, Y); 173(L, A, C, D, F, H, K, N, W, Y); 174(N, D, G,H, I, L, P, S, T, V); 181(R, A, C, E, F, I, L, M, N, Q, S, T, V, Y);183(H, A, C, D, E, F, L, M, N, P, Q, V, W, Y); 211(P, A, C, D, F, K, L,M, N, Q, S, V); 222(V, A, D, E, F, G, H, I, L, M, N, R, S, T, Y); 251(N,A, C, G, H, K, L, P, Q, R, S, T, V, W, Y); 262(F, A, C, D, E, G, H, K,L, M, Q, R, S, Y); 273(G, C, D, E, H, K, L, M, P, Q, S, T, V, Y); 303(S,A, C, D, E, G, L, M, Q, R); 311(N, D, E, F, G, H, K, L, M, Q, R, T, Y);314(N, A, D, E, G, H, I, K, L, M, Q, S, T, V, Y); 320(R, A, D, E, H, K,M, N, Q, S, T, Y); 323(S, A, C, D, E, F, G, H, I, L, M, P, R, T, V, Y);345(E, A, G, H, K, L, M, N, Q, S, T, Y); 346(E, A, C, D, G, H, K, M, N,Q, R, S, T, V, Y); 360(E, A, C, F, H, K, L, N, P, Q, R, T, V, Y); 361(Q,A, C, D, E, G, H, S, T, V, W); 375(P, A, D, E, G, H, I, K, M, Q, R, T,V, Y); 388(P, A, C, D, F, G, H, K, L, N, Q, R, S, T, V, Y); 391(E, A, C,G, H, I, K, L, N, R, S, W); 395(K, A, D, E, G, M, Q, R, S, T, V); 400(K,A, F, G, H, I, L, M, Q, T, V, W); 408(H, E, G, K, M, N, P, Q, R, S, T);416(E, A, D, F, G, H, K, L, N, Q, R, T, V, W, Y); 418(N, A, D, I, K, L,M, Q, S, T, V); 419(T, D, E, H, K, L, M, N, P, Q, R, S, W, Y); 420(A, D,F, G, H, I, L, M, Q, R, S, T, V, W); 421(H, A, C, D, E, I, K, L, M, N,R, V, W, Y); 422(P, A, C, E, F, G, L, M, T, V, Y); 423(N, C, D, E, F, H,I, L, R, S, T); 424(S, A, C, D, E, G, I, N, Q, T, V, W); 434(A, C, D, E,F, H, I, K, M, N, P, Q, R, S, T, V); 435(G, A, C, E, K, M, N, P, Q, R,T); 452(S, A, C, E, F, H, K, N, Q, T, W, Y); 458(R, C, D, E, H, K, L, M,N, S, T, V, Y); 459(T, A, C, D, E, G, H, L, N, P, S); 461(T, A, C, D, E,F, G, K, L, N, P, Q, R, V, Y); and 466(A, D, E, G, K, N, P, Q, R, S).

The productive positions in Amy 707 that fall within the previouslydescribed Productivity Scores of “3 and 4” and the substitutions withinthose positions that are combinable are listed below. Position numberingis based on the mature Amy707 protein listed in SEQ ID NO: 3.

1(H, A, C, E, F, I, K, L, M, N, Q, R, T, W); 2(H, A, C, D, E, F, G, I,K, L, M, N, P, Q, S, W); 3(N, A, C, D, E, F, K, L, M, Q, S, T, V); 4(G,D, E, F, H, I, K, L, M, P, S, T, W); 5(T, A, C, D, G, H, I, M, N, Q, S,V, W); 6(N, A, E, G, Q, S, T); 7(G, A, D, H, I, L, M, P, Q, R, S, T, V,Y); 16(Y, A, D, E, H, N, T, W); 17(L, A, D, G, S, T, V); 20(D, A, C, E,G, H, I, N, S, Y); 22(N, E, G, I, L, M, Q, R, S, T, V, W); 25(N, A, C,G, K, M, S, T, V, Y); 28(N, A, C, D, E, G, H, K, Q, R, W, Y); 29(S, A,C, D, E, F, H, K, M, N, R, T, V, W, Y); 32(S, C, D, E, G, I, L, M, N, Q,R, W, Y); 33(N, C, D, H, I, K, M, Q, R, T, V, W, Y); 35(K, A, C, E, F,G, H, I, L, M, N, Q); 41(A, C, D, I, K, M, Q, S); 54(N, A, C, D, E, F,G, M, Q, S, V, W); 70(N, C, D, E, F, G, H, I, K, L, M, R, S, V); 73(G,D, E, K, M, Q, R, S, T, W, Y); 81(T, A, C, D, F, I, K, N, P, S); 82(R,A, C, F, I, K, M, Q, S, V, Y); 84(Q, C, D, E, F, K, L, M, N); 86(Q, E,H, I, K, R, T, V, W, Y); 87(A, D, K, M, T); 91(S, A, E, H, K, M, N, Q,R, T, V); 94(N, A, C, D, F, G, H, K, L, M, Q, R); 95(N, A, C, D, F, G,H, I, Q, R, S, T, Y); 98(Q, A, C, D, E, G, H, K, R); 113(A, C, E, F, G,H, I, K, M, R, V, Y); 116(M, A, C, D, E, F, G, I, L, N, P, Q, R, T, V,W); 117(V, E, L, P, R, S, T); 118(R, D, E, G, L, Q, T, V, W); 125(N, C,F, G, H, I, L, M, R, S, T, W, Y); 133(G, A, D, H, P, Q, S, T); 136(T, C,D, F, G, K, L, M, N, P, Q, R, Y); 142(R, C, E, F, G, H, K, S, T, Y);144(D, E, I, K, M, S, Y); 146(P, A, C, D, E, F, G, H, M, R, S, W, Y);149(G, A, C, D, E, F, H, K, L, P, R, V, W); 151(T, D, E, G, H, I, L, M,Q, V); 160(Y, A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S); 169(Q, A,C, D, E, F, G, H, I, K, M, N, S, V, W, Y); 172(R, A, C, E, G, H, M, Q,S, Y); 173(L, A, C, D, F, H, K, N, W, Y); 174(N, D, G, H, I, L, P, S, T,V); 175(N, A, D, E, L, M, R, S); 181(R, A, C, E, F, I, L, M, N, Q, S, T,V, Y); 183(H, A, C, D, E, F, L, M, N, P, Q, V, W, Y); 210(H, A, C, D, E,M, N, Q, R, S); 211(P, A, C, D, F, K, L, M, N, Q, S, V); 215(N, D, F, K,L, M, Q); 216(E, A, C, G, H, L, N, Q, S, T, Y); 218(R, A, C, E, F, H, K,M); 219(N, A, C, D, M, R, T); 222(V, A, D, E, F, G, H, I, L, M, N, R, S,T, Y); 233(F, A, C, H, L, M, W, Y); 251(N, A, C, G, H, K, L, P, Q, R, S,T, V, W, Y); 258(G, D, I, K, M, N, P, R, S, V); 259(K, A, C, D, E, G, H,P, Q, R, T); 261(M, A, C, E, G, I, Q, T); 262(F, A, C, D, E, G, H, K, L,M, Q, R, S, Y); 273(G, C, D, E, H, K, L, M, P, Q, S, T, V, Y); 286(H, A,C, E, F, L, M, N, T, V); 299(N, G, H, M, R, S, T, Y); 303(S, A, C, D, E,G, L, M, Q, R); 311(N, D, E, F, G, H, K, L, M, Q, R, T, Y); 314(N, A, D,E, G, H, I, K, L, M, Q, S, T, V, Y); 319(Q, A, D, E, G, H, N, R, Y);320(R, A, D, E, H, K, M, N, Q, S, T, Y); 323(S, A, C, D, E, F, G, H, I,L, M, P, R, T, V, Y); 326(V, A, C, H, M, N, T); 337(E, A, C, D, N, Q, S,T, Y); 345(E, A, G, H, K, L, M, N, Q, S, T, Y); 346(E, A, C, D, G, H, K,M, N, Q, R, S, T, V, Y); 360(E, A, C, F, H, K, L, N, P, Q, R, T, V, Y);361(Q, A, C, D, E, G, H, S, T, V, W); 363(Y, A, D, E, I, K, M, N, Q, V,W); 372(Y, H, I, K, M, Q, R, T, V); 375(P, A, D, E, G, H, I, K, M, Q, R,T, V, Y); 379(V, A, I, L, M, N, Q, R, S, Y); 388(P, A, C, D, F, G, H, K,L, N, Q, R, S, T, V, Y); 389(I, E, F, G, L, M, Q, S, V); 391(E, A, C, G,H, I, K, L, N, R, S, W); 394(Q, C, D, E, G, H, L, R, V, Y); 395(K, A, D,E, G, M, Q, R, S, T, V); 400(K, A, F, G, H, I, L, M, Q, T, V, W); 402(N,C, I, K, L, S, V, Y); 408(H, E, G, K, M, N, P, Q, R, S, T); 416(E, A, D,F, G, H, K, L, N, Q, R, T, V, W, Y); 418(N, A, D, I, K, L, M, Q, S, T,V); 419(T, D, E, H, K, L, M, N, P, Q, R, S, W, Y); 420(A, D, F, G, H, I,L, M, Q, R, S, T, V, W); 421(H, A, C, D, E, I, K, L, M, N, R, V, W, Y);422(P, A, C, E, F, G, L, M, T, V, Y); 423(N, C, D, E, F, H, I, L, R, S,T); 424(S, A, C, D, E, G, I, N, Q, T, V, W); 433(G, A, C, D, E, K, M, N,R); 434(A, C, D, E, F, H, I, K, M, N, P, Q, R, S, T, V); 435(G, A, C, E,K, M, N, P, Q, R, T); 437(S, A, C, D, K, N, T); 445(N, A, C, E, G, K, Q,R, T); 446(K, A, C, F, H, M, Q, S, T, Y); 450(V, A, E, I, L, Q, R, S,T); 452(S, A, C, E, F, H, K, N, Q, T, W, Y); 454(I, A, C, F, L, M, S,V); 458(R, C, D, E, H, K, L, M, N, S, T, V, Y); 459(T, A, C, D, E, G, H,L, N, P, S); 460(G, E, H, K, N, Q, S); 461(T, A, C, D, E, F, G, K, L, N,P, Q, R, V, Y); 466(A, D, E, G, K, N, P, Q, R, S); 471(N, C, D, E, H, Q,R, Y); 477(G, A, D, K, N, P, Q, R, T); 483(V, C, G, H, M, R, S, T);484(N, A, E, G, H, Q, R, S); and 485(K, H, M, P, Q, S, T).

The productive positions in Amy 707 that fall within the previouslydescribed Productivity Scores of “2, 3 and 4” and the substitutionswithin those positions that are combinable are listed below. Positionnumbering is based on the mature Amy707 protein listed in SEQ ID NO: 3.

1(H, A, C, E, F, I, K, L, M, N, Q, R, T, W); 2(H, A, C, D, E, F, G, I,K, L, M, N, P, Q, S, W); 3(N, A, C, D, E, F, K, L, M, Q, S, T, V); 4(G,D, E, F, H, I, K, L, M, P, S, T, W); 5(T, A, C, D, G, H, I, M, N, Q, S,V, W); 6(N, A, E, G, Q, S, T); 7(G, A, D, H, I, L, M, P, Q, R, S, T, V,Y); 16(Y, A, D, E, H, N, T, W); 17(L, A, D, G, S, T, V); 20(D, A, C, E,G, H, I, N, S, Y); 22(N, E, G, I, L, M, Q, R, S, T, V, W); 23(H, F, M,Q, T); 25(N, A, C, G, K, M, S, T, V, Y); 26(R, K, Q, T); 27(L, A, I, V);28(N, A, C, D, E, G, H, K, Q, R, W, Y); 29(S, A, C, D, E, F, H, K, M, N,R, T, V, W, Y); 30(D, E, M, N, Q, R); 32(S, C, D, E, G, I, L, M, N, Q,R, W, Y); 33(N, C, D, H, I, K, M, Q, R, T, V, W, Y); 35(K, A, C, E, F,G, H, I, L, M, N, Q); 36(S, D, G, K, Q, T); 41(A, C, D, I, K, M, Q, S);47(A, G, M, P, S); 50(G, C, S); 52(S, K, L, M, R, T); 54(N, A, C, D, E,F, G, M, Q, S, V, W); 56(V, E, N, S); 63(L, M, N, Q); 68(E, A, D, Q);70(N, C, D, E, F, G, H, I, K, L, M, R, S, V); 73(G, D, E, K, M, Q, R, S,T, W, Y); 74(T, G, S); 77(T, A, I, N, S, V); 81(T, A, C, D, F, I, K, N,P, S); 82(R, A, C, F, I, K, M, Q, S, V, Y); 83(S, K, M, N, Q, R, T);84(Q, C, D, E, F, K, L, M, N); 86(Q, E, H, I, K, R, T, V, W, Y); 87(A,D, K, M, T); 89(V, A, C, I); 90(T, G, M, Q, R, S); 91(S, A, E, H, K, M,N, Q, R, T, V); 94(N, A, C, D, F, G, H, K, L, M, Q, R); 95(N, A, C, D,F, G, H, I, Q, R, S, T, Y); 96(G, D, E, N); 98(Q, A, C, D, E, G, H, K,R); 99(V, A, C, I); 100(Y, C, F, I); 103(V, A, C, F, I, L, T); 110(G, A,P, S); 113(A, C, E, F, G, H, I, K, M, R, V, Y); 116(M, A, C, D, E, F, G,I, L, N, P, Q, R, T, V, W); 117(V, E, L, P, R, S, T); 118(R, D, E, G, L,Q, T, V, W); 123(N, A, C, L); 125(N, C, F, G, H, I, L, M, R, S, T, W,Y); 128(N, C, E, L, Y); 133(G, A, D, H, P, Q, S, T); 134(E, D, P, S, T,V); 135(Y, C, F, L, M, Q); 136(T, C, D, F, G, K, L, M, N, P, Q, R, Y);138(E, D, L, M, N); 139(A, C, G); 142(R, C, E, F, G, H, K, S, T, Y);144(D, E, I, K, M, S, Y); 146(P, A, C, D, E, F, G, H, M, R, S, W, Y);147(G, A, D, I, L); 149(G, A, C, D, E, F, H, K, L, P, R, V, W); 150(N,H, L, M, P, R, S); 151(T, D, E, G, H, I, L, M, Q, V); 154(S, L, R, Y);156(K, A, D, S); 158(R, A, C, K, L, N, Q); 160(Y, A, C, D, E, F, G, H,I, K, L, M, N, P, Q, R, S); 169(Q, A, C, D, E, F, G, H, I, K, M, N, S,V, W, Y); 170(S, A, C, E, K); 171(R, M, S, T); 172(R, A, C, E, G, H, M,Q, S, Y); 173(L, A, C, D, F, H, K, N, W, Y); 174(N, D, G, H, I, L, P, S,T, V); 175(N, A, D, E, L, M, R, S); 179(K, C, L, M, Q); 181(R, A, C, E,F, I, L, M, N, Q, S, T, V, Y); 183(H, A, C, D, E, F, L, M, N, P, Q, V,W, Y); 184(G, A, C, D, E, L, N); 186(A, D, E, G, N); 195(N, F, L, W, Y);206(I, C, H, M, N, S, T); 210(H, A, C, D, E, M, N, Q, R, S); 211(P, A,C, D, F, K, L, M, N, Q, S, V); 215(N, D, F, K, L, M, Q); 216(E, A, C, G,H, L, N, Q, S, T, Y); 218(R, A, C, E, F, H, K, M); 219(N, A, C, D, M, R,T); 222(V, A, D, E, F, G, H, I, L, M, N, R, S, T, Y); 225(T, A, K, R);226(N, A, D, E, K, Y); 227(T, A, E, K); 229(G, C, F, V); 231(D, E, G,T); 233(F, A, C, H, L, M, W, Y); 235(I, L, M, V); 244(S, D, E, H, N, Q);247(R, A, E, L, T); 249(W, F, L, M); 250(I, L, M, V); 251(N, A, C, G, H,K, L, P, Q, R, S, T, V, W, Y); 252(H, D, E, K, N); 257(T, A, M, S);258(G, D, I, K, M, N, P, R, S, V); 259(K, A, C, D, E, G, H, P, Q, R, T);260(N, D, K, P, R); 261(M, A, C, E, G, I, Q, T); 262(F, A, C, D, E, G,H, K, L, M, Q, R, S, Y); 273(G, C, D, E, H, K, L, M, P, Q, S, T, V, Y);280(Q, D, H, K, M, N, V); 285(N, E, K, M, S, T); 286(H, A, C, E, F, L,M, N, T, V); 287(S, A, D, N, Y); 288(V, C, I, L, T); 298(Y, F, R, W);299(N, G, H, M, R, S, T, Y); 302(K, C, E, M, Q, R, S, T); 303(S, A, C,D, E, G, L, M, Q, R); 304(G, C, K, R, S, V); 306(N, A, D, G); 311(N, D,E, F, G, H, K, L, M, Q, R, T, Y); 312(I, L, M, V); 314(N, A, D, E, G, H,I, K, L, M, Q, S, T, V, Y); 318(V, C, I, L, M, S, T); 319(Q, A, D, E, G,H, N, R, Y); 320(R, A, D, E, H, K, M, N, Q, S, T, Y); 323(S, A, C, D, E,F, G, H, I, L, M, P, R, T, V, Y); 324(H, A, C, K, M, Y); 326(V, A, C, H,M, N, T); 337(E, A, C, D, N, Q, S, T, Y); 339(A, G, S, T); 341(E, A, D,F, G, H, K, Y); 345(E, A, G, H, K, L, M, N, Q, S, T, Y); 346(E, A, C, D,G, H, K, M, N, Q, R, S, T, V, Y); 351(L, A, C, M, Q); 355(L, I, K, M,V); 356(T, C, I, L, Q, V); 357(L, A, H, M); 358(T, A, C, G, I,; 359(R,I, V, W); 360(E, A, C, F, H, K, L, N, P, Q, R, T, V, Y); 361(Q, A, C, D,E, G, H, S, T, V, W); 363(Y, A, D, E, I, K, M, N, Q, V, W); 364(P, A, C,G); 365(S, A, G, N, V); 366(V, C, I, L); 368(Y, G, L, M, Q); 372(Y, H,I, K, M, Q, R, T, V); 374(I, C, N, Q, S); 375(P, A, D, E, G, H, I, K, M,Q, R, T, V, Y); 377(H, A, G, K, M, T); 378(G, E, H, L, M, N); 379(V, A,I, L, M, N, Q, R, S, Y); 380(P, D, E, G, H, K, Q, S); 381(A, G, N, Q, R,S, T); 387(D, E, G, N); 388(P, A, C, D, F, G, H, K, L, N, Q, R, S, T, V,Y); 389(I, E, F, G, L, M, Q, S, V); 391(E, A, C, G, H, I, K, L, N, R, S,W); 392(A, C, G, S); 394(Q, C, D, E, G, H, L, R, V, Y); 395(K, A, D, E,G, M, Q, R, S, T, V); 396(Y, K, M, N); 400(K, A, F, G, H, I, L, M, Q, T,V, W); 402(N, C, I, K, L, S, V, Y); 405(L, A, C, M, N, T, V); 406(D, A,C, L, N, Q); 408(H, E, G, K, M, N, P, Q, R, S, T); 413(W, F, H, I, L,Y); 414(T, A, C, S, V); 415(R, C, W, Y); 416(E, A, D, F, G, H, K, L, N,Q, R, T, V, W, Y); 418(N, A, D, I, K, L, M, Q, S, T, V); 419(T, D, E, H,K, L, M, N, P, Q, R, S, W, Y); 420(A, D, F, G, H, I, L, M, Q, R, S, T,V, W); 421(H, A, C, D, E, I, K, L, M, N, R, V, W, Y); 422(P, A, C, E, F,G, L, M, T, V, Y); 423(N, C, D, E, F, H, I, L, R, S, T); 424(S, A, C, D,E, G, I, N, Q, T, V, W); 426(L, A, N, S); 430(M, G, I, L, V); 433(G, A,C, D, E, K, M, N, R); 434(A, C, D, E, F, H, I, K, M, N, P, Q, R, S, T,V); 435(G, A, C, E, K, M, N, P, Q, R, T); 436(G, A, C, D, Q, S); 437(S,A, C, D, K, N, T); 438(K, C, E, H, S); 439(W, H, L, M, Q); 441(F, H, N,Y); 444(R, A, K, Q); 445(N, A, C, E, G, K, Q, R, T); 446(K, A, C, F, H,M, Q, S, T, Y); 450(V, A, E, I, L, Q, R, S, T); 452(S, A, C, E, F, H, K,N, Q, T, W, Y); 454(I, A, C, F, L, M, S, V); 457(N, G, H, Q, R, T);458(R, C, D, E, H, K, L, M, N, S, T, V, Y); 459(T, A, C, D, E, G, H, L,N, P, S); 460(G, E, H, K, N, Q, S); 461(T, A, C, D, E, F, G, K, L, N, P,Q, R, V, Y); 463(T, E, K, L, P, Q, R); 465(N, D, G, Q); 466(A, D, E, G,K, N, P, Q, R, S); 471(N, C, D, E, H, Q, R, Y); 476(G, D, E, H, N, R);477(G, A, D, K, N, P, Q, R, T); 481(I, L, T, V); 483(V, C, G, H, M, R,S, T); 484(N, A, E, G, H, Q, R, S); and 485(K, H, M, P, Q, S, T).

The productive positions in Amy 707 that fall within the previouslydescribed Productivity Scores of “1, 2, 3 and 4” and the substitutionswithin those positions that are combinable are listed below. Positionnumbering is based on the mature Amy707 protein listed in SEQ ID NO: 3.

1(H, A, C, E, F, I, K, L, M, N, Q, R, T, W); 2(H, A, C, D, E, F, G, I,K, L, M, N, P, Q, S, W); 3(N, A, C, D, E, F, K, L, M, Q, S, T, V); 4(G,D, E, F, H, I, K, L, M, P, S, T, W); 5(T, A, C, D, G, H, I, M, N, Q, S,V, W); 6(N, A, E, G, Q, S, T); 7(G, A, D, H, I, L, M, P, Q, R, S, T, V,Y); 10(M, I, L); 12(Y, A); 16(Y, A, D, E, H, N, T, W); 17(L, A, D, G, S,T, V); 18(P, A, E); 19(N, D, L); 20(D, A, C, E, G, H, I, N, S, Y); 22(N,E, G, I, L, M, Q, R, S, T, V, W); 23(H, F, M, Q, T); 25(N, A, C, G, K,M, S, T, V, Y); 26(R, K, Q, T); 27(L, A, I, V); 28(N, A, C, D, E, G, H,K, Q, R, W, Y); 29(S, A, C, D, E, F, H, K, M, N, R, T, V, W, Y); 30(D,E, M, N, Q, R); 31(A, S); 32(S, C, D, E, G, I, L, M, N, Q, R, W, Y);33(N, C, D, H, I, K, M, Q, R, T, V, W, Y); 34(L, F, M); 35(K, A, C, E,F, G, H, I, L, M, N, Q); 36(S, D, G, K, Q, T); 40(T, K, N); 41(A, C, D,I, K, M, Q, S); 47(A, G, M, P, S); 50(G, C, S); 52(S, K, L, M, R, T);53(Q, A); 54(N, A, C, D, E, F, G, M, Q, S, V, W); 56(V, E, N, S); 61(Y,F); 63(L, M, N, Q); 64(Y, H); 66(L, M, V); 68(E, A, D, Q); 70(N, C, D,E, F, G, H, I, K, L, M, R, S, V); 72(K, R); 73(G, D, E, K, M, Q, R, S,T, W, Y); 74(T, G, S); 75(V, I, M); 77(T, A, I, N, S, V); 81(T, A, C, D,F, I, K, N, P, S); 82(R, A, C, F, I, K, M, Q, S, V, Y); 83(S, K, M, N,Q, R, T); 84(Q, C, D, E, F, K, L, M, N); 86(Q, E, H, I, K, R, T, V, W,Y); 87(A, D, K, M, T); 88(A, M); 89(V, A, C, I); 90(T, G, M, Q, R, S);91(S, A, E, H, K, M, N, Q, R, T, V); 93(K, H, R); 94(N, A, C, D, F, G,H, K, L, M, Q, R); 95(N, A, C, D, F, G, H, I, Q, R, S, T, Y); 96(G, D,E, N); 97(I, V); 98(Q, A, C, D, E, G, H, K, R); 99(V, A, C, I); 100(Y,C, F, I); 101(G, A); 103(V, A, C, F, I, L, T); 110(G, A, P, S); 111(A,S); 112(D, C, E); 113(A, C, E, F, G, H, I, K, M, R, V, Y); 115(E, Q);116(M, A, C, D, E, F, G, I, L, N, P, Q, R, T, V, W); 117(V, E, L, P, R,S, T); 118(R, D, E, G, L, Q, T, V, W); 119(A, C, S); 122(V, C); 123(N,A, C, L); 124(P, N, T); 125(N, C, F, G, H, I, L, M, R, S, T, W, Y);126(N, D); 128(N, C, E, L, Y); 129(Q, V); 132(T, S); 133(G, A, D, H, P,Q, S, T); 134(E, D, P, S, T, V); 135(Y, C, F, L, M, Q); 136(T, C, D, F,G, K, L, M, N, P, Q, R, Y); 138(E, D, L, M, N); 139(A, C, G); 140(W, F,Y); 142(R, C, E, F, G, H, K, S, T, Y); 144(D, E, I, K, M, S, Y); 145(F,M, Y); 146(P, A, C, D, E, F, G, H, M, R, S, W, Y); 147(G, A, D, I, L);149(G, A, C, D, E, F, H, K, L, P, R, V, W); 150(N, H, L, M, P, R, S);151(T, D, E, G, H, I, L, M, Q, V); 153(S, N); 154(S, L, R, Y); 155(F,W); 156(K, A, D, S); 158(R, A, C, K, L, N, Q); 160(Y, A, C, D, E, F, G,H, I, K, L, M, N, P, Q, R, S); 162(F, M); 165(V, C, T); 167(W, F, M);168(D, C); 169(Q, A, C, D, E, F, G, H, I, K, M, N, S, V, W, Y); 170(S,A, C, E, K); 171(R, M, S, T); 172(R, A, C, E, G, H, M, Q, S, Y); 173(L,A, C, D, F, H, K, N, W, Y); 174(N, D, G, H, I, L, P, S, T, V); 175(N, A,D, E, L, M, R, S); 176(R, K, T); 178(Y, W); 179(K, C, L, M, Q); 181(R,A, C, E, F, I, L, M, N, Q, S, T, V, Y); 182(G, C, D); 183(H, A, C, D, E,F, L, M, N, P, Q, V, W, Y); 184(G, A, C, D, E, L, N); 186(A, D, E, G,N); 195(N, F, L, W, Y); 196(G, C); 203(Y, N); 206(I, C, H, M, N, S, T);210(H, A, C, D, E, M, N, Q, R, S); 211(P, A, C, D, F, K, L, M, N, Q, S,V); 215(N, D, F, K, L, M, Q); 216(E, A, C, G, H, L, N, Q, S, T, Y);217(L, M); 218(R, A, C, E, F, H, K, M); 219(N, A, C, D, M, R, T); 221(G,I, V); 222(V, A, D, E, F, G, H, I, L, M, N, R, S, T, Y); 225(T, A, K,R); 226(N, A, D, E, K, Y); 227(T, A, E, K); 228(L, I, M); 229(G, C, F,V); 230(L, M); 231(D, E, G, T); 233(F, A, C, H, L, M, W, Y); 235(I, L,M, V); 238(V, I); 243(Y, F); 244(S, D, E, H, N, Q); 245(F, E, M); 247(R,A, E, L, T); 249(W, F, L, M); 250(I, L, M, V); 251(N, A, C, G, H, K, L,P, Q, R, S, T, V, W, Y); 252(H, D, E, K, N); 253(V, M); 257(T, A, M, S);258(G, D, I, K, M, N, P, R, S, V); 259(K, A, C, D, E, G, H, P, Q, R, T);260(N, D, K, P, R); 261(M, A, C, E, G, I, Q, T); 262(F, A, C, D, E, G,H, K, L, M, Q, R, S, Y); 263(A, S); 265(A, G, S); 273(G, C, D, E, H, K,L, M, P, Q, S, T, V, Y); 276(E, C, T); 280(Q, D, H, K, M, N, V); 283(N,D, G); 285(N, E, K, M, S, T); 286(H, A, C, E, F, L, M, N, T, V); 287(S,A, D, N, Y); 288(V, C, I, L, T); 292(P, L, M); 296(N, Q); 297(L, M);298(Y, F, R, W); 299(N, G, H, M, R, S, T, Y); 301(S, A, G); 302(K, C, E,M, Q, R, S, T); 303(S, A, C, D, E, G, L, M, Q, R); 304(G, C, K, R, S,V); 306(N, A, D, G); 307(Y, A, F); 310(R, Q, S); 311(N, D, E, F, G, H,K, L, M, Q, R, T, Y); 312(I, L, M, V); 313(F, M, Y); 314(N, A, D, E, G,H, I, K, L, M, Q, S, T, V, Y); 317(V, L); 318(V, C, I, L, M, S, T);319(Q, A, D, E, G, H, N, R, Y); 320(R, A, D, E, H, K, M, N, Q, S, T, Y);321(H, W, Y); 322(P, D); 323(S, A, C, D, E, F, G, H, I, L, M, P, R, T,V, Y); 324(H, A, C, K, M, Y); 326(V, A, C, H, M, N, T); 327(T, L);328(F, V); 329(V, I); 334(S, T); 337(E, A, C, D, N, Q, S, T, Y); 339(A,G, S, T); 341(E, A, D, F, G, H, K, Y); 343(F, T, Y); 344(V, C, I);345(E, A, G, H, K, L, M, N, Q, S, T, Y); 346(E, A, C, D, G, H, K, M, N,Q, R, S, T, V, Y); 347(W, A, D); 350(P, E); 351(L, A, C, M, Q); 352(A,S); 354(A, S); 355(L, I, K, M, V); 356(T, C, I, L, Q, V); 357(L, A, H,M); 358(T, A, C, G, I,; 359(R, I, V, W); 360(E, A, C, F, H, K, L, N, P,Q, R, T, V, Y); 361(Q, A, C, D, E, G, H, S, T, V, W); 363(Y, A, D, E, I,K, M, N, Q, V, W); 364(P, A, C, G); 365(S, A, G, N, V); 366(V, C, I, L);367(F, Y); 368(Y, G, L, M, Q); 369(G, A, S); 372(Y, H, I, K, M, Q, R, T,V); 374(I, C, N, Q, S); 375(P, A, D, E, G, H, I, K, M, Q, R, T, V, Y);377(H, A, G, K, M, T); 378(G, E, H, L, M, N); 379(V, A, I, L, M, N, Q,R, S, Y); 380(P, D, E, G, H, K, Q, S); 381(A, G, N, Q, R, S, T); 382(M,K); 386(I, L, V); 387(D, E, G, N); 388(P, A, C, D, F, G, H, K, L, N, Q,R, S, T, V, Y); 389(I, E, F, G, L, M, Q, S, V); 390(L, M, V); 391(E, A,C, G, H, I, K, L, N, R, S, W); 392(A, C, G, S); 394(Q, C, D, E, G, H, L,R, V, Y); 395(K, A, D, E, G, M, Q, R, S, T, V); 396(Y, K, M, N); 397(A,G); 400(K, A, F, G, H, I, L, M, Q, T, V, W); 401(Q, H, M); 402(N, C, I,K, L, S, V, Y); 403(D, E, T); 405(L, A, C, M, N, T, V); 406(D, A, C, L,N, Q); 408(H, E, G, K, M, N, P, Q, R, S, T); 410(I, N); 411(I, V);412(G, A, S); 413(W, F, H, I, L, Y); 414(T, A, C, S, V); 415(R, C, W,Y); 416(E, A, D, F, G, H, K, L, N, Q, R, T, V, W, Y); 417(G, A); 418(N,A, D, I, K, L, M, Q, S, T, V); 419(T, D, E, H, K, L, M, N, P, Q, R, S,W, Y); 420(A, D, F, G, H, I, L, M, Q, R, S, T, V, W); 421(H, A, C, D, E,I, K, L, M, N, R, V, W, Y); 422(P, A, C, E, F, G, L, M, T, V, Y); 423(N,C, D, E, F, H, I, L, R, S, T); 424(S, A, C, D, E, G, I, N, Q, T, V, W);425(G, A); 426(L, A, N, S); 427(A, C, T); 428(T, N, S); 429(I, M);430(M, G, I, L, V); 431(S, A, C); 433(G, A, C, D, E, K, M, N, R); 434(A,C, D, E, F, H, I, K, M, N, P, Q, R, S, T, V); 435(G, A, C, E, K, M, N,P, Q, R, T); 436(G, A, C, D, Q, S); 437(S, A, C, D, K, N, T); 438(K, C,E, H, S); 439(W, H, L, M, Q); 441(F, H, N, Y); 442(V, A, C); 444(R, A,K, Q); 445(N, A, C, E, G, K, Q, R, T); 446(K, A, C, F, H, M, Q, S, T,Y); 448(G, F, N); 450(V, A, E, I, L, Q, R, S, T); 451(W, F); 452(S, A,C, E, F, H, K, N, Q, T, W, Y); 454(I, A, C, F, L, M, S, V); 457(N, G, H,Q, R, T); 458(R, C, D, E, H, K, L, M, N, S, T, V, Y); 459(T, A, C, D, E,G, H, L, N, P, S); 460(G, E, H, K, N, Q, S); 461(T, A, C, D, E, F, G, K,L, N, P, Q, R, V, Y); 463(T, E, K, L, P, Q, R); 465(N, D, G, Q); 466(A,D, E, G, K, N, P, Q, R, S); 467(D, E); 469(W, Y); 471(N, C, D, E, H, Q,R, Y); 473(S, P); 474(V, S); 475(N, D, E); 476(G, D, E, H, N, R); 477(G,A, D, K, N, P, Q, R, T); 478(S, A, G); 479(V, T); 481(I, L, T, V);482(W, Y); 483(V, C, G, H, M, R, S, T); 484(N, A, E, G, H, Q, R, S); and485(K, H, M, P, Q, S, T).

What is claimed is:
 1. A variant α-amylase polypeptide derived from aparental α-amylase polypeptide, comprising at least two combinablemutations at productive amino acid positions; wherein the mutations areat position S452 and R142, wherein the variant has at least 99% aminoacid sequence identity with SEQ ID NO:
 3. 2. The variant amylase ofclaim 1, wherein the mutations are S452Y and R142K.
 3. The variantamylase of claim 1, further comprising a deletion corresponding to aresidue selected from the group consisting of Arg-181, Gly-182, His-183,and Gly-184, using SEQ ID NO: 3 for numbering.
 4. The variant amylase ofclaim 1, further comprising deletions corresponding to residues Arg-181and Gly-182, using SEQ ID NO: 3 for numbering.
 5. A compositioncomprising the variant amylase of claim
 1. 6. The composition of claim5, wherein the composition is effective for removing starchy stains fromlaundry, dishes, or textiles.
 7. The composition of claim 5, furthercomprising a surfactant.